Human Molecular Genetics, 2001, Vol. 10, No. 6 605-616
© 2001 Oxford University Press
PTEN inhibits insulin-stimulated MEK/MAPK activation and cell growth by blocking IRS-1 phosphorylation and IRS-1/Grb-2/Sos complex formation 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 3 January 2001; Revised and Accepted 31 January 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene PTEN encodes a dual-specificity phosphatase that recognizes protein substrates and phosphatidylinositol-3,4,5-triphosphate. PTEN seems to play multiple roles in tumour suppression and the blockade of phosphoinositide-3-kinase signalling is important for its growth suppressive effects, although precise mechanisms are not fully understood. In this study, we show that PTEN plays a unique role in the insulin-signalling pathway in a breast cancer model. Ectopic expression of wild-type PTEN in MCF-7 epithelial breast cancer cells resulted in universal inhibition of Akt phosphorylation in response to stimulation by diverse growth factors and selective inhibition of MEK/extracellular signal-regulated kinase (ERK) phosphorylation stimulated by insulin or insulin-like growth factor 1 (IGF-1). The latter was accompanied by a decrease in the phosphorylation of insulin receptor substrate 1 (IRS-1) and the association of IRS-1 with Grb2/Sos, without affecting the phosphorylation status of the insulin receptor and Shc, nor Shc/Grb2 complex formation. The MEK inhibitor, PD980059, but not the PI3K inhibitor, wortmannin, abolished the effect of PTEN on insulin-stimulated cell growth. Without addition of insulin, wortmannin reduced PTEN-mediated growth suppression, whereas PD980059 had little effect, suggesting that PTEN suppresses insulin-stimulated cell growth by blocking the mitogen-activated protein kinase (MAPK) pathway. Furthermore, PD980059 treatment led to the downregulation of cyclin D1 and the suppression of cell cycle progression. Our data suggest that PTEN blocks MAPK phosphorylation in response to insulin stimulation by inhibiting the phosphorylation of IRS-1 and IRS-1/Grb2/Sos complex formation, which leads to downregulation of cyclin D1, inhibition of cell cycle progression and suppression of cell growth.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene PTEN/MMAC1/TEP1 (1–3) has been implicated in a variety of human cancers and three inherited harmatoma tumour syndromes: Cowden syndrome, which has a high risk of breast, thyroid and other cancers (4–6), Bannayan–Riley–Ruvalcaba syndrome (7,8) and a Proteus-like syndrome (9). PTEN, a dual-specificity phosphatase, seems to play multiple roles in tumour suppression. Ectopic expression of PTEN suppresses tumourigenicity and cell growth (10,11) through G1 cell cycle arrest (12,13), apoptosis (13,14) or both (15–17). Numerous studies have established the essential role of inhibition of the phosphoinositide-3-kinase (PI3K) signalling pathway in PTEN-mediated growth suppression. In Caenorhabditis elegans, the PTEN homologue daf-18 has been shown to play a distinct role in an insulin-like receptor signalling pathway (18–22).
Like other polypeptide growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), insulin and insulin-like growth factors (IGFs) simultaneously activate both mitogen-activated protein kinase (MAPK) and PI3K after binding and triggering autophosphorylation of the receptors. Unlike most activated receptor tyrosine kinases, which provide direct SH2 recognition sites, activated insulin receptors associate with and phosphorylate a family of adapter proteins, including insulin receptor substrate 1 (IRS-1) (23), IRS-2 (24), Gab1 and Dos (25).
IRS-1 contains 20–22 potential tyrosine phosphorylation sites (26) and becomes heavily phosphorylated upon activation of the insulin receptor or IGF receptor (27–29). Phosphorylated IRS-1 provides binding sites for several SH2-containing enzymes and adapter proteins, including Grb-2, PI3K, Syp (also known as SH-PTP/PTP1C), Fyn, Nck, and Crk (25,30). The binding of PI3K, through its SH2-containing regulatory subunit, to IRS leads to its activation (31–33). The recruitment of Grb-2 and Sos to IRS-1 leads to the activation of the guanine nucleotide exchange factor Sos (34,35), which facilitates the exchange of guanosine 5'-diphosphate (GDP) for guanosine 5'-triphosphate (GTP) in Ras, which in turn activates Ras and triggers the Ras/Raf/MEK/MAPK signalling cascade (36–38). Insulin-stimulated MAPK activation also occurs through Shc, independent of IRS-1 (35,39). However, the effect of PTEN on growth factor-stimulated MAPK activation in the existing literature remains controversial (14,40,41).
IRS-1 is also heavily phosphorylated on serine/threonine at baseline, both of which can increase further upon insulin stimulation (26). It contains over 30 potential serine/threonine phosphorylation sites in motifs recognized by various kinases, such as PI3K (42–44), Akt (45), casein kinase (46,47), MAPK (48), glycogen synthase kinase 3 (GSK3) (49) and protein kinase Cs (PKCs) (50,51). The role of serine/threonine phosphorylation in IRS-1 signalling is controversial as it may down-regulate IRS signalling by inhibiting tyrosine phosphorylation (25,43), but there are reports which present opposing data (45).
Given the fact that so many potential phosphorylation sites in IRS and the activated IRS signalling complex contain signalling molecules covering diverse pathways, the exact nature of downstream signals engaged by IRS-1 depends on the stochiometry of phosphorylation that reflects a balance between the action of kinases and phosphatases. We hypothesized that PTEN modulates IRS signalling by interacting with the upstream kinases or directly dephosphorylating IRS. Taking together the genetic evidence from C.elegans, it is conceivable that PTEN might regulate insulin/IGF-stimulated cell growth through modulating the IRS/Grb2/Sos-activated Raf-MAPK pathway in combination with its negative regulatory effect on the PI3K pathway. We sought to investigate this possibility in MCF-7 breast cancer epithelial cells and found that PTEN specifically blocks insulin-stimulated MAPK phosphorylation through inhibition of IRS-1 phosphorylation and IRS-1/Grb-2/Sos complex formation, which leads to the downregulation of cyclin D1 and subsequent inhibition of cell cycle progression, and the suppression of cell growth.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PTEN inhibits the phosphorylation of MEK and ERKs
Activation of MAPKs plays an essential role in many cellular functions. Phosphorylation is required for the activation of MAPKs. The MAPK family members include ERK1/2, stress activated protein kinase (SAPK)/Jun N-terminal kinases (JNKs), p38 group and ERK3, -4 and -5. To investigate the role of PTEN in the regulation of the activity of these MAPKs, we analysed the phosphorylation status of the MAPKs in a breast cancer cell line model system expressing wild-type PTEN or the C124S phosphatase-dead mutant by western blot, using an antibody specifically against the phosphorylated forms of the MAPKs (Fig. 1). Induction of wild-type PTEN in MCF-7 cells resulted in a reduction in the phosphorylation of Akt and p44/42 ERK1/2 (Fig. 1A, third and top panels; MCF-7/PTEN.wt/Tet–) compared with controls (MCF-7/PTEN.wt/Tet+). Conversely, over-expression of the C124S phosphatase-dead mutant resulted in an increase in Akt phosphorylation, without affecting the phosphorylation of p44/42 ERK1/2 compared with cells grown in the presence of tetracycline.
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Mitogen-activated protein kinase kinases (MAPKKs), including MEK1/2 and MKK3, -4, -5, -6 and -7, are dual specificity kinases that phosphorylate MAPKs. MEK1 and -2 are the kinases known only to activate ERKs by phosphorylation. The kinase activity of MEK1/2 is also regulated by upstream kinases through phosphorylation. To see whether the effect of PTEN on MAPK phosphorylation was due to inhibition of the activity of the MEKs, phosphorylation of MEK1/2 was examined by western blot using an anti-phospho-MEK1/2-specific antibody. The antibody recognizes two species with apparent molecular weight of
40–45 kDa. The anti-MEK1 and -MEK2 antibodies detected a band corresponding to the small and large species, respectively. The patterns of MEK1/2 phosphorylation were similar to those of ERKs, i.e. ectopic expression of wild-type PTEN resulted in decreased phosphorylated MEK1/2 (Fig. 1B). The reduction in the phosphorylation of ERK1/2 and MEK1/2 caused by over-expression of PTEN was not the result of a reduction in the absolute amounts of MAPK protein (Fig. 1A, panel 2) or MEK1 and MEK2 protein (Fig. 1B, panels 2 and 3). MEK1/2 can be phosphorylated and activated by several upstream kinases, including Raf, Mos and MEK kinases (MEKKs). The serine/threonine kinases serve as a central intermediate connecting upstream tyrosine kinases and Ras with downstream MEK. Mos is expressed only in embryonic tissue (52) and unlikely to play a major role in MEK activation in general (53). Some MEKKs can phosphorylate MEK as well as SAPK/JNKs and p38 through MKKs (54). To investigate whether PTEN also has an effect on the phosphorylation of other members of the MAPK family, we analysed the phosphorylation of SAPK/JNKs and p38 (Fig. 1C). Stimulation of the cells with insulin significantly increased the phosphorylation levels of MEK1/2 (Fig. 1C, top panel) and p44/42 ERKs (panel 2), without affecting phosphorylation of ether SAPK/JNK or p38 (Fig. 1C, panels 3 and 4). Over-expression of wild-type PTEN significantly decreased the insulin-stimulated phosphorylation of MEK1/2 and ERK1/2, whereas over-expression of the C124S mutant had no effect. In contrast to the suppression of ERK1/2 phosphorylation, over-expression of PTEN did not inhibit the phosphorylation of SAPK and P38. A slight increase in the basal phosphorylation of p38 and SAPK (both 46 and 54 kDa species) appeared with induction of PTEN (Fig. 1C, Control/Tet– of panels 3 and 4). Osmotic stress induced by 0.7 M NaCl strongly stimulated the phosphorylation of MEK1/2, p38 and SAPK/JNKs, but no difference between Tet+ and Tet– cultures was observed.
PTEN specifically blocks insulin-stimulated phosphorylation of ERKs
Our observations that PTEN blocked MEK phosphorylation without effect on the phosphorylation of SAPK and p38 suggest that inhibition of PTEN-mediated phosphorylation of the ERKs may be through Ras/Raf, the central intermediates connecting peptide growth factors and cytokines to MAPK activation. To see whether PTEN could play a general role in the negative regulation of growth factor-stimulated MAPK activation, we examined the effect of PTEN over-expression on MAPK phopsphorylation in response to several peptide growth factors known to activate Ras through Grb2/Sos, such as 1–0ieoyl-lysophosphatidic acid (LPS), which activates Ras through G protein-coupled receptors, and TPA, the tumour promoting reagent which activates MAPK through PKC (Fig. 2). Interestingly, expression of PTEN universally blocked Akt phosphorylation in response to insulin, IGF-1, EGF, PDGF, fibroblast growth factor (FGF) and LPS (Fig. 2A, all Tet– lanes). In contrast, PTEN specifically inhibited insulin- and IGF-stimulated MAPK (i.e. ERK1/2) phosphorylation without effect on the MAPK phosphorylation in response to PDGF, EGF, FGF and LPS (Fig. 2A). PTEN also had no effect on MAPK phosphorylation stimulated by phorbol-13-myristrate-13-acetate (PMA), the latter of which strongly stimulates MAPK phosphorylation in MCF-7 cells. These data suggest that PTEN inhibits insulin-stimulated MAPK by affecting the components upstream of Ras.
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24 h. In contrast, there is no time dependency associated with PTEN mediated inhibition of Akt phosphorylation.While we have observed that PTEN inhibited insulin- and IGF-stimulated MAPK (ERK1/2) phosphorylation, we wished to explore whether this effect was dependent on time of insulin exposure (Fig. 2B). PTEN inhibition of insulin-stimulated phosphorylation of ERK1/2 became evident only at 30 min of insulin exposure and persisted through 24 h. As a control, we noted that PTEN inhibition of Akt phosphorylation was not dependent on insulin exposure time.
The effect of PTEN on Shc phosphorylation and its association with the Grb2 complex
Stimulation of the insulin receptor could lead to the activation of the Ras/Raf/MEK/MAPK pathway through phosphorylation of the adapter protein Shc. Phosphorylated Shc provides docking sites for the Grb-2/Sos complex, which in turn activates Ras. It has been reported that over-expression of PTEN in U-87MG glioma cells blocks EGF-stimulated MAPK phosphorylation by dephosphorylation of Shc. To see whether this is the case in our system, we examined the effect of PTEN on Shc phosphorylation and its association with Grb-2. Shc has three isoforms, 66, 52 and 46 kDa (Fig. 3B), due to different translational start sites (55); only the 52 kDa isoform was shown to be phosphorylated in response to insulin or EGF stimulation (Fig. 3A). After 10 min of stimulation with insulin, increased phosphorylation of Shc and increased amounts of Grb-2 in the anti-Shc immunoprecipitate were observed (Fig. 3A, P-Shc and Grb2). Both of these returned to basal levels at 30 min, when a strong phosphorylation of Shc and Shc/Grb-2 association were observed, suggesting that insulin can only induce weak and transient Shc phosphorylation and Shc/Grb-2 association. In the presence of insulin or EGF, PTEN had no effect on either Shc phosphorylation or Shc/Grb-2 association.
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Effect of PTEN on insulin receptor ß phosphorylation
Binding of insulin to insulin receptor leads to the phosphorylation of the ß-chain and activation of its intrinsic kinase. The activated kinase phosphorylates its downstream targets and triggers several signalling pathways, including phosphorylation of MAPK. To investigate whether PTEN inhibits insulin-stimulated MAPK phophorylation by affecting insulin receptor phosphorylation, the phosphorylation of insulin receptor was examined by immunoprecipitation of insulin receptor using an anti-insulin ß-chain antibody, followed by western blot with 4G10 antibody, which recognizes proteins phosphorylated on tyrosine. As shown in Figure 4, stimulation of cells with insulin markedly increased the phosphorylation of insulin receptor, but there was no difference between immunoprecipitated insulin receptor from PTEN over-expressing cells and controls (Fig. 4, panel 1). The same blot was re-probed with an anti-IR ß antibody (Fig. 4, panel 2), confirming that equal amounts of IR ß were immunoprecipitated.
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PTEN inhibits insulin-stimulated IRS-1/Grb-2/Sos complex formation
It has been established that the activation of insulin receptor by insulin leads to IRS-1 phosphorylation. Phosphorylated IRS-1 then acts as the nucleus in recruiting other molecules to the complex, including the p85 subunit of PI3K, tyrosine phosphatase SH-PTP or Syp and small adaptor proteins Nck and Grb-2. The SH3 domain of Grb-2 directs the association with Sos, the guanylnucleotide exchange factor for Ras, and subsequently activates the Ras/MAPK pathway. We therefore examined the possibility that the effect of PTEN on insulin-stimulated MAPK phosphorylation might be through alteration of IRS-1/Grb-2/Sos complex formation. As Figure 5A shows, in the absence of insulin, inmmunoprecipitation of IRS-1 from PTEN over-expressing cells and control cells with IRS-1-specific antibody resulted in the co-immunoprecipitation of essentially identical amounts of Grb2 and Sos, as detected by anti-Grb2 and anti-Sos antibodies. Insulin stimulation increased the amounts of Grb-2 and Sos co-immuniprecipitated with IRS-1 from control cells. However, the Grb-2 and Sos co-immunoprecipitated with IRS-1from PTEN over-expressing cells were reduced compared with controls, although slightly increased compared with unstimulated cells. Interestingly, the immunoprecipitated IRS-1 from insulin-stimulated cells had a decreased electrophoretic mobility, and this mobility shift was clearly affected by the absence or presence of PTEN (Fig. 5A).
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Having observed that PTEN altered the amounts of Grb-2 and Sos co-immunoprecipitated with IRS-1, we next examined the effect of PTEN on the association of IRS-1 with Grb-2 by Grb-2 immunoprecipitation (Fig. 5B). Consistent with the results shown in Figure 5A, over-expression of PTEN led to a reduction in the amount of IRS-1 and Sos co-immunoprecipitated with Grb-2 following insulin stimulation (Fig. 5B). Thus, PTEN over-expression seems to have prevented the association of IRS-1 with Grb-2/Sos.
PTEN inhibits the insulin-stimulated mobility shift of IRS-1
IRS-1 contains 20–22 tyrosine phosphorylation sites and over 30 potential serine/threonine phosphorylation sites. Upon insulin stimulation, IRS-1 has been shown to become heavily phosphorylated at tyrosine residues and also phosphorylated at serine/threonine residues (26). To determinine whether the IRS-1 mobility shift, which appears to be affected by PTEN (Fig. 5A, bottom panel) was due to the insulin-induced phosphorylation of IRS-1, we isolated IRS-1 by immunoprecipitation from control and PTEN over-expressing cells in the presence or absence of insulin stimulation for 10 and 30 min as well as EGF stimulation for 30 min. After exposure or non-exposure with insulin or EGF, and in the presence or absence of PTEN over-expression, immunoprecipitated IRS-1 was subjected to electrophoresis through low cross-linking SDS–PAGE and transferred to nitrocellulose membranes. IRS-1 was visualized after blotting with anti-IRS-1 antibody and tyrosine phosphorylation was detected by a 4G10 antibody. As shown in Figure 6A, stimulation of cells with insulin for 10 min dramatically increased IRS-1 phosphorylation on tyrosine without clearly affecting electrophoretic mobility. In contrast, insulin stimulation for 30 min markedly reduced the IRS-1 electrophoretic mobility without further increasing its tyrosine phosphorylation. Over-expression of PTEN did not affect insulin-stimulated tyrosine phosphorylation of IRS-1 but clearly blocked the insulin-induced mobility shift. EGF had no effect on either tyrosine phosphorylation or electrophoretic mobility of IRS-1. Treatment of immunoprecipitated IRS-1 from insulin-stimulated cells with alkaline phosphatase abrogated the IRS-1 mobility shift [Fig. 6B, calf intestinal alkaline phosphatase (CIP)], suggesting that the decrease in IRS-1 mobility induced by insulin was due to an increase in phosphorylation. Taken together with the facts that PTEN has no effect on tyrosine phosphorylation of IRS-1, and PTEN reversed the insulin-stimulated IRS-1 mobility shift, these observations suggest that PTEN blocks insulin-stimulated phosphorylation of IRS-1 on serine/threonine.
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PTEN inhibits insulin-stimulated cell growth by blocking the MAPK pathway
Having shown that PTEN dephosphorylates IRS-1 and prevents IRS-1/Grb-2/Sos complex formation and MAPK phosphorylation in response to insulin, we next investigated inhibition of the MAPK and PI3K pathways by PTEN in insulin-stimulated cell growth. Manipulations were performed under low growth factor culture conditions (Fig. 7, Ch.S). As Figure 7 shows, treatment of cells with the PI3K inhibitor, wortmannin, led to a reduction of cell number by 50% irrespective of whether insulin was present or not (Tet+/I+wort versus Tet+/Ch.S+wort) and inhibition of the MAPK pathway by the MAPK inhibitor PD980059 significantly retarded insulin-stimulated cell growth (Tet+/I+PD versus Tet+/I), but had only a slight effect on cell growth without the addition of insulin (Tet+/Ch.S+PD versus Tet+/Ch.S). Induction of PTEN expression under low growth factor culture conditions (Tet–/Ch.S versus Tet+/Ch.S) resulted in a 48.6% growth suppression. The growth suppression associated with PTEN over-expression was reduced to 26.9% when wortmannin was added (Tet–/Ch.S+wort versus Tet+/Ch.S+wort) and to 40.7% in the presence of PD980059 (Tet–/Ch.S+PD versus Tet–/Ch.S+PD). Addition of insulin to the culture medium significantly reduced the PTEN-mediated growth suppression from 48.6 to 27.4% (Fig. 7, column 4). In the presence of insulin, blocking MAPK almost completely abrogated PTEN-mediated growth suppression (Fig. 7; I + PD, 8.6% of growth inhibition), whereas inhibition of PI3K increased the growth suppressive effect of PTEN (Tet–/I+wort). PD980059 completely blocked insulin-stimulated MAPK phosphorylation without affecting Akt phosphorylation (Fig. 8B). These data suggested that the inhibition of the MAPK and PI3K pathways by PTEN plays a different role in cell growth in response to insulin and other growth factors in serum, and that PTEN inhibits insulin-stimulated cell growth mainly through blocking the MAPK pathway.
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MEK inhibitor mimics the effect of PTEN on cell cycle progression and cyclin D1 expression
The MAPK pathway plays an essential role in the regulation of growth factor-stimulated cyclin D expression and cell cycle progression. We have found that over-expression of PTEN in MCF-7 cells causes G1 arrest (15) and reduced cyclin D1 protein levels (L.-P. Weng and C. Eng, unpublished data). Having shown that PTEN inhibits insulin-stimulated cell growth through blocking MAPK, we further investigated whether the blockade of MAPK signalling by PTEN could lead to the inhibition of cell cycle progression and cyclin D1 expression (Fig. 8). Induction of PTEN expression in the presence of insulin led to an increase in the G1 population from 47.6 to 60% (Fig 8A, Tet+ versus Tet–). The MAPK inhibitor had a similar effect, increasing the G1 population from 47.6 to 62.1% (Fig 8A, PD+ versus PD–). Induction of PTEN expression in the presence of insulin resulted in reduction of Akt and MAPK phosphorylation as well as cyclin D1 levels (Fig. 8B). Inhibition of MAPK by PD980059 had similar effects on cyclin D1 levels and MAPK phosphorylation as that of PTEN, but without affecting Akt phosphorylation (Fig. 8B). These data suggest that blocking MAPK is necessary and sufficient for PTEN to inhibit cyclin D1 expression and cell cycle progression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It has been well established that MAPK plays an essential role in cell cycle progression. Here, we demonstrate that over-expression of PTEN in the well-documented MCF-7 breast cancer line inhibits cell growth by blocking the MAPK pathway in a phosphatase activity-dependent manner. Several lines of evidence from this study suggest that PTEN inhibits the phosphorylation of ERKs through the IRS-1/Grb-2/Sos-mediated signalling pathway (Fig. 9). First, over-expression of PTEN blocked the phosphorylation of ERK1/2 in response to insulin or IGF-1 but not to many other stimuli, including osmotic stress and several inflammatory cytokines. The non-response to stimuli other than insulin or IGF-1 is germane for the following reasons. The inflammatory cytokine LPS is known to activate the MEK kinases, which can in turn phosphorylate MEK1/2, SAPK/JNK and p38. The tumour promoter PMA activates Raf, and EGF and basic FGF (bFGF) can activate Ras via Grb-2/Sos. Taken together, therefore, these data suggest that none of the components from Grb-2 to ERK is likely to be the direct target of PTEN. Second, while the inhibition of insulin-stimulated MAPK phosphorylation could be the result of a decrease in insulin receptor phosphorylation, we have shown that this is not the case since PTEN per se did not affect insulin receptor phosphorylation. Third, activation of insulin receptor could activate the Ras-MAPK pathway in an IRS-1-independent manner through phosphorylating Shc, which then recruits Grb2 and Sos to the activated receptor complex. However, Gu et al. (40) reported that over-expression of PTEN in U-87MG glioma cells inhibits integrin- and growth factor (PDGF and EGF)-mediated MAPK activation through the dephosphorylation of Shc without affecting the phosphorylation of Akt, consequently suppressing cell spreading but not cell growth. Apparent conflicting results have been reported as well: over-expression of PTEN using retroviral vectors in the same U-87MG line (41) or in U251 glioma cells (14) resulted in inhibition of cell growth secondary to blockade of the PI3K/Akt pathway without affecting MAPK phosphorylation or EGF-stimulated MAPK phosphorylation. Our results clearly show that Shc is not the target of PTEN, at least in MCF-7, since the phosphorylation of Shc and its association with Grb-2 in response to the stimulation with either insulin or EGF were not affected by over-expression of PTEN. In other words, PTEN had no effect on the IRS-1-independent activation of the Ras-Raf-MAPK pathway. Furthermore, Shc-mediated Ras-MAPK activation is a common pathway for receptor tyrosine kinases, and PTEN did not have an effect on EGF- or bFGF-stimulated MAPK phosphorylation. In addition, we have demonstrated that insulin-stimulated MAPK phosphorylation persisted for at least 24 h and a clear effect of PTEN on insulin-stimulated MAPK phosphorylation already occurred at 30 min. Stimulation with insulin produced only mild and transient Shc phosphorylation and Shc/Grb-2 association, both of which returned to basal levels at 30 min, when insulin-stimulated increases in IRS-1 phosphorylation and IRS-1/Grb-2 association persisted. These data in toto suggest that Shc/Grb2 interaction mediates transient MAPK phosphorylation whereas IRS-1/Grb-2 interaction predominates in sustained MAPK phosphorylation in response to insulin, and PTEN affects only the sustained MAPK phosphorylation mediated by the IRS-1/Grb-2 interaction. The role of Shc and IRS-1 in regulating the Ras-MAPK pathway must be cell-type dependent. In skeletal muscle, the IRS-1/Grb-2 pathway predominates (56). In the 32D myeloid progenitor cell line, both pathways, Shc/Grb-2 and IRS-1/Grb-2, contribute under certain conditions (25,34). Binding of Grb-2/Sos to Shc may be the major mechanism in liver (56), L6 rat myoblasts and Chinese hamster ovary cells (57).
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Finally, we have demonstrated that over-expression of PTEN results in the suppression of IRS-1/Grb-2/Sos complex formation (Fig. 9). The impaired IRS-1/Grb-2/Sos complex stimulated by insulin in PTEN over-expressing cells is most likely due to decreases in IRS-1 phosphorylation. Insulin stimulation resulted in marked increases in tyrosine phosphorylation and mobility shift. Stripping phosphorylation by CIP abolished the IRS-1 mobility shift, suggesting that the insulin-induced mobility shift was due to phosphorylation. It has been shown that the increase in phosphophorylation of IRS-1 on serine/threonine obtained after treatment with the serine/threonine phosphatase inhibitor, okadaic acid, resulted in a decrease in electrophoretic mobility (58). Interestingly, we have shown that insulin stimulation for 10 min markedly increased tyrosine phosphorylation, but no obvious decrease in IRS-1 electrophoretic mobility was detected; a clear mobility shift occurred at 30 min after insulin stimulation but no further increases in tyrosine phosphorylation were observed. PTEN significantly suppressed the insulin-stimulated IRS-1 mobility shift without altering IRS-1 phosphorylation on tyrosine. Our results, therefore, suggest a differential two-phase phosphorylation of IRS-1 in response to insulin stimulation: tyrosine phosphorylation occurs initially followed by a delayed serine/threonine phosphorylation. PTEN only affects the later phase of phosphorylation, i.e. the phosphorylation on serine/threonine.
It has been demonstrated that phosphorylation on Tyr-895 is essential for IRS-1 interacting with the SH2 domain of Grb-2 (58). To our knowledge, the effect of serine/threonine phosphorylation on IRS-1/Grb-2 association has not yet been reported. Given that PTEN blocks insulin-stimulated IRS-1 phosphorylation on serine/threonine and suppresses insulin-induced IRS-1/Grb-2/Sos complex formation, the phosphorylation on certain serine/threonine residues might also be important for the interaction of IRS-1 with Grb-2/Sos and subsequent activation of the Ras/Raf/Mek/Erk pathway in response to insulin. However, whether there are other pathways between the IRS-1/Grb-2/Sos and MAPK pathways in the context of PTEN is as yet unknown and requires further investigation. We failed to demonstrate the physical interaction between PTEN and IRS-1 by immunoprecipation-western blot, so whether PTEN directly dephosphorylates IRS-1 or inhibits its upstream kinases remains to be further investigated. Although MAPK (48), Akt (45) and PI3K (42–44) can phosphorylate IRS-1, it is unlikely to be the case in MCF-7 breast cancer cells, since PTEN has no effect on the kinase activity of PI3K (59) and no effect on the binding of PI3K to IRS-1. As a negative control, EGF stimulation strongly increased Akt and MAPK phosphorylation, but had no effect on the IRS-1 mobility shift.
The MAPK pathway plays an essential role in the regulation of mitogen-induced cyclin D expression (60–63) and cell cycle progression (64–67). Consistent with these observations, over-expression of PTEN inhibits MAPK phosphorylation, cyclin D1 expression and cell cycle progression in response to insulin stimulation. MEK inhibitor PD980059 mimics all those effects. Moreover, inhibition of MAPK activity by PD980059 in the presence of insulin diminished PTEN’s growth suppressive effect; in contrast, without the addition of insulin, PD980059 had little effect. This little effect may reflect the cell growth contributed by insulin or IGFs present in serum. These results suggest that inhibition of MAPK phosphorylation is necessary and sufficient for PTEN to suppress cell growth stimulated by insulin, and that PTEN may affect a different signalling pathway to suppress cell growth in response to exposure to growth factors other than insulin.
There is little doubt that the PI3K pathway also plays an important role in transmission of signals from various mitogens to cell proliferation. Numerous studies have demonstrated that PTEN suppresses cell growth by blocking the PI3K/Akt signalling pathway (12–15,41,68–70). In agreement with these studies, our data show that over-expression of PTEN blocks Akt phosphorylation in response to all the stimuli we have tested, including insulin, IGF1, EGF, bFGF, LPS and PMA, as long as the factors can stimulate Akt phosphorylation. Induction of PTEN expression in the presence of serum without addition of insulin, the PI3K inhibitor wortmannin strongly, but not completely, reversed the effect of PTEN on cell growth. The incomplete effect of wortmannin on PTEN-mediated growth suppression may be partially due to the stability of this reagent and relatively low concentrations used due to the toxicity at high doses. In the presence of insulin, wortmannin did not inhibit, but instead, enhanced the growth suppressive effect of PTEN. The enhancement may be due, in this instance, to the profound contribution of the MAPK pathway to cell growth when the PI3K pathway is suppressed. These results together suggest that PTEN’s suppression of cell growth stimulated by growth factors other than insulin is mediated by blockade of the PI3K pathway; furthermore, the inhibition of this pathway is not relevant to the effect between PTEN on insulin-mediated cell growth.
In summary, PTEN exerts its growth suppressive effect through the inhibition of two separate signalling pathways, the MAPK pathway and the PI3K pathway, depending on the cell signalling context. It has been demonstrated that the PI3K pathway, but not the MAPK pathway, plays an important role in the regulation of insulin-mediated metabolism, such as glucose uptake (30,71). Taken together with the genetic evidence from C.elegans (19,22), our results suggest that PTEN might coordinate the integrity of insulin action through interaction with both the PI3K pathway and the MAPK pathway.
As part of this study, we decided to examine the insulin-IRS pathway in a breast cancer cell line model because of emerging data attempting to associate insulin signalling, IGF receptors (IGFRs) and IRSs in breast carcinogenesis. In one report, Schnarr et al. (72) report on an association between downregulation of IGFR-I and IRS-1 and high proliferative rates and worse prognosis in human breast carcinomas. There is little doubt that PTEN plays a role in inherited susceptibility to breast cancer (8,73) and sporadic breast cancer (74–76). It would also appear that somatic mutation and deletion in PTEN have been associated with more advanced cancers, e.g. those of the prostate and brain (77,78). In the presence of insulin, PTEN seemed to prevent IRS-1/Sos/Grb-2 complex formation and thus, signalling down that particular pathway(s), an observation that differs from PTEN over-expression in the absence of exogenous growth factors. Thus, the relationship between PTEN and the insulin-IRS-1 pathway in breast carcinogenesis is complex and might be modulated depending on the micro-environmental milieu.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
The MCF-7 breast cancer cells expressing wild-type and phosphatase-dead mutant C124S PTEN were generated as described previously (15). The C124 position is a highly conserved residue in the phosphatase core motif of PTEN. We have chosen this mutant because missense and nonsense mutations affecting C124 have been described in Cowden syndrome/Bannayan–Riley–Ruvalcaba syndrome and in sporadic tumours (8,73). The C124S mutant has been formally demonstrated to be phosphatase dead (69).
The MCF-7/Toff cell line (Clontech) and PTEN stable expressing clones were maintained in Dulbecco’s modified Eagle’s Medium/10% fetal bovine serum (FBS) (Gibco BRL Life Technologies) with 100 U/ml penicillin G (Sigma), 100 µg/ml streptomycin sulfate (Sigma; similar media plus 100 µg/ml geneticin and 1 µg/ml tetracycline).
Induction of PTEN expression
Sub-confluent stock cells were washed twice with phosphate-buffered saline (PBS), trypsinized by phenol red-free typsin-EDTA (Gibco BRL) and diluted at a 1:3 ratio into estrogen-free culture media, containing MEBM (Clontech; phenol red-free), 5% charcoal/Dextran-treated FBS (HyClone, UT) and 1 µg/ml tetracycline for 3 days. Then equal numbers of cells were plated into the same medium without tetracycline to induce PTEN expression and into medium containing 1 µg/ml tetracycline (Tet+) as a control and cultured for 48 h, unless otherwise noted.
Cell growth assay
Cell growth was measured by methylene blue. Equal numbers of cells were plated into Tet+ and Tet– media in 12-well plates and cultured for 48 h. After incubation, medium was removed and cells were washed with PBS and fixed with 12.5% glutaraldehyde (Fisher) for 20 min at room temperature. Cells were rinsed with distilled water and incubated with 0.05% methylene blue (Sigma) for 30 min, again rinsed with water and then incubated with 800 µl of 0.33 M HCl for 30 min to extract the methylene blue. 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. When cells were to be treated with PD980059 or wortmannin, cells were plated into the media containing each of these reagents at concentrations indicated in the Results section and media containing fresh reagents were added every 24 h. A similar amount of dimethylsulfoxide (DMSO) (vehicle) was added to control cells. All experiments were performed in triplicate and a mean and standard deviation presented.
Fluorescence-activated cell sorter (FACS) analysis
At the end of incubation, cells were trypsinized and washed into ice-cold PBS. Cells were fixed by adding them drop-wise into ice-cold 80% ethanol while vortexing, followed by incubation on ice 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 µg/ml propidium iodine (Sigma) and 5 µg/ml RNase A (New England Biolab). DNA content was determined by FACS scan analysis (Becton Dickinson). All FACS assays were performed in triplicate.
Protein extraction and immunoblotting
After PTEN induction, cells were washed twice with ice-cold PBS and lysed in cold lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 5 µM PMSF, 5 µg/ml leupeptin, pepstatin A and aprotinin, 1 mM Na3VO4, 2 mM NaF, 2 mM Na4PO7 and 10 mM ß-glycerophsphate) for 10 min on ice. Insoluble material was removed from cell lysates by centrifugation at 4°C. Protein concentration was calculated using the Bradford reagent. The Bradford reagent and other chemicals were purchased from Sigma. Cell lysates were mixed with equal volumes of 2x Laemmli sample buffer, boiled for 10 min, resolved by 10% SDS–PAGE and transferred onto nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in TBST (10 mM Tris–HCL pH 8.0, 100 mM NaCl and 0.05% Tween-20) or TBST with 5% BSA for 1 h at room temperature, and then incubated with appropriate primary antibody for 2 h at room temperature or overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody (Promega) at 1:5000 dilution for 1 h at room temperature. Protein signals were detected by enhanced chemiluminesence (Amersham).
For growth factor stimulation, cells were starved by exposure to serum-free medium for 24 h before adding growth factor(s). Insulin, IGF-1, b-FGF, PDGF and EGF were purchased from Gibco BRL. Wortmannin and PMA-EGF were purchased from Sigma. PD590089 and LPS and EGF were purchased from New England Biolab and Cayman Chemical, respectively. The anti-PTEN monoclonal antibody 6H2.1 was raised agaist the C-terminal of PTEN (76). The polyclonal anti-phospho-Akt, anti-Akt, anti-phospho-MAPK, anti-MAPK, anti-phospho-MEK1/2, anti-phospho-SAPK and anti-phospho-p38 (New England Biolab) were used at 1:1000 dilution; polyclonal anti-IRS-1, anti-IRS-2, anti-IRß, anti-Sos1/2, anti-MEK1 and anti-MEK2 (Santa Cruz Biotechology) at 1:250 dilution; 4G10 and polyclonal anti-p85 PI3K (Upstate Biotechnology) at 1 µg/ml and monoclonal anti-
-tubulin (Sigma), at 1:5 000 dilution. Monoclonal and polyclonal anti-Shc, monoclonal anti-Grb2 (Transduction Laboratory) were used at 1, 0.1, 0.5 and 1 µg/ml, respectively.
Immunoprecipitation
After PTEN induction, cells were washed twice with ice-cold PBS and lysed in cold lysis buffer. After removal of insoluble material, the supernatant was precleaned with protein A/agarose (Santa Cruz) or anti-mouse IgG/agarose (Sigma). Five hundred millilitres of the cell lysates (500 g protein/ml) were incubated with 2.5 g monoclonal or polyclonal antibodies for 2 h or overnight at 4°C followed by 10 µl of packed protein A/agarose or anti-mouse IgG/agarose for 2 h. The immunoprecipitates were washed three times with lysis buffer and then resuspended in Laemmli sample buffer. The immunoprecipitates were boiled for 5 min, centrifuged, and the protein in the supernatant was resolved by SDS–PAGE.
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ACKNOWLEDGEMENTS |
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This work is partially funded by the American Cancer Society (RPG98-211-01CCE) to C.E, the Susan G. Komen Breast Cancer Research Foundation (BCTR 2000 462) to C.E. and the National Cancer Institute (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 Program, 420 West 12th Avenue, Suite 690 Tzagournis MRF, Columbus, OH 43210, USA. Tel: +1 614 292 2347; Fax: +1 614 688 3582; Email: eng-1@medctr.osu.edu
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REFERENCES |
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