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Article
Nature Medicine  8, 1136 - 1144 (2002)
Published online: 16 September 2002; | doi:10.1038/nm762

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer

Giuseppe Viglietto1, Maria Letizia Motti1, Paola Bruni2, Rosa Marina Melillo1, Amelia D'Alessio3, Daniela Califano3, Floriana Vinci4, Gennaro Chiappetta3, Philip Tsichlis5, Alfonso Bellacosa6, 7, Alfredo Fusco1 & Massimo Santoro1

1 Istituto di Endocrinologia ed Oncologia Sperimentale, CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Naples, Italy

2 Dipartimento di Biochimica e Biotecnologie Mediche, Naples, Italy

3 Istituto Nazionale dei Tumori "Fondazione Pascale", Naples, Italy

4 Dipartimento di Chimica Organica e Biochimica, Complesso Universitario Monte S. Angelo, Naples, Italy

5 Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania, USA

6 Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

7 Department of Medical Genetics, Catholic University, Medical School, 00168 Rome, Italy

Correspondence should be addressed to Giuseppe Viglietto viglietto@sun.ceos.na.cnr.it
The cyclin-dependent kinase inhibitor p27kip1 is a putative tumor suppressor for human cancer. The mechanism underlying p27kip1 deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27kip1-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27kip1, is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27kip1 in the cytoplasm, precluding p27kip1-induced G1 arrest. Conversely, the p27kip1-T157A mutant accumulates in cell nuclei and Akt does not affect p27kip1−T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27kip1 accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27kip1, secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27kip1 are functionally inactivated and the proliferation of breast cancer cells is sustained.
Disruption of cell-cycle control1, 2 is a hallmark of cancer3. Most human cancer cells bear mutations that affect the retinoblastoma susceptibility gene product pRb and/or the tumor suppressor gene p53 either by disabling these genes directly or by targeting genes that act epistatically to prevent their function4. D- and E-type cyclins are overexpressed in several human cancers5, 6, 7, 8. Other tumors lack the cyclin-dependent kinase (CDK) inhibitors p15ink4A, p16ink4B and p18ink4C consequent to mutations, deletions and/or promoter methylation9, 10.

By contrast, mutations in genes encoding the Kip/Cip inhibitors p21cip1 and p27kip1 are rare11. That notwithstanding, in mice p27kip1 behaves like a tumor suppressor: the loss of only one gene copy increases susceptibility to cancer12. Up to 50% of human tumors lack p27kip1 expression mainly because of accelerated proteolysis13, 14, 15. Low p27kip1 level is associated with reduced survival in cancer patients7, 13, 15.

Paradoxically, some tumors may contain elevated concentrations of p27kip1 protein8, 13, 14, 15, suggesting they have evolved other mechanisms to circumvent p27kip1 growth inhibition. Accordingly, in tumors with abundant p27kip1 expression, the protein is often mislocalized to the cytoplasm8, 16, 17. Because the growth-restraining activity of p27kip1 depends on its nuclear localization, the aberrant compartmentalization of p27kip1 probably impairs its function8, 18, 19, 20, 21. Although proteins such as tuberin19, Jab1 (ref. 22) and D-type cyclins8 have been involved in the cytoplasm-to-nucleus shuttling of p27kip1, the pathways that regulate p27kip1 localization in tumor cells are unknown.

The serine/threonine protein kinase Akt (here termed 'Akt')/protein kinase B (PKB) plays a pivotal role in tumorigenesis23. It affects the growth and survival of cancer cells through the phosphorylation and relocalization of key regulatory molecules24 such as Bad (ref. 25), caspase-9 (ref. 26), AFX and FKHRL1 (refs. 27,28) and p21cip1 (ref. 29). Akt is a downstream effector of phosphoinositide-3-kinase (PI3K)30. Amplification of PI3K and Akt genes, loss-of-function mutations of PTEN (which negatively regulates the PI3K-Akt pathway)31 or gain-of-function mutations of upstream Akt activators (for example, oncogenic Ras and receptor tyrosine kinases) are features of human cancer24. In breast cancer, the PI3K-Akt pathway is a critical downstream effector of the HER2/ErbB2, insulin-like growth factor receptor (IGFR) and epidermal growth factor receptor (EGFR)32, 33, 34, 35. Here we show that Akt-dependent phosphorylation and mislocalization of p27kip1 is a mechanism whereby p27kip1 is functionally inactivated in human breast cancer.

PI3K-Akt regulates growth and p27kip1 in breast cancer
PI3K-Akt blockade with LY294002 (20 muM), an inhibitor of the catalytic subunit of PI3K, for 24 hours induced accumulation of breast cancer cells (MDA-MB468 and MCF-7) in G1 phase (Fig. 1a). In parallel, LY294002 suppressed Akt phosphorylation and moderately increased p27kip1 protein levels (data not shown). The most marked effect of LY294002 was accumulation of p27kip1 in cell nuclei (Fig. 1b and c, lane 5). Conversely, blockade of the MEK-Erk pathway with PD98059 (50 muM for 24 h) or U0126 (5 muM for 24 h) did not affect cell growth (Fig. 1a and data not shown) or p27kip1 localization (Fig. 1b and c, lane 6). Similar results were obtained in two additional breast cancer cell lines (MDA-MB-231, MDA-MB-436) (data not shown). Consequent to the nuclear accumulation of p27kip1 upon PI3K-Akt blockade, the p27kip1 fraction complexed with nuclear CDK2 increased, thereby inhibiting (by about 90%) CDK2-mediated phosphorylation of histone H1 (Fig. 1d).

Figure 1. PI3K-Akt inhibition induces G1 arrest of breast cancer cells and nuclear accumulation of p27kip1.
Figure 1 thumbnail

a, Flow-cytometric analysis of MCF-7 and MDA-MB468 cells transiently transfected with CMV or myrAkt after treatment with LY294002 (LY), PD98059 (PD) or solvent. *, fEGFP-positive cells; , EGFP-negative cells. Within bars: , G2/M; shaded square, S; , G1. b, Immunoblot analysis of PI3K- or MEK-Erk-dependent p27kip1 subcellular localization in MCF-7 cells. Cells were treated with DMSO (lanes 1 & 4), LY294002 (lanes 2 & 5) or PD98059 (lanes 3 & 6). c, Immunoblot analysis of PI3K- or MEK-Erk-dependent p27kip1 subcellular localization in MDA-MB468 cells. Cells were treated with DMSO (lanes 1 & 4), LY294002 (lanes 2 & 5) or PD98059 (lanes 3 & 6). d, Immunoblot analysis of Akt-dependent p27kip1 subcellular localization and activity in MDA-MB468 cells. Cells were transfected with CMV (lanes 1, 2, 5 & 6), myrAkt (lanes 3 & 7) or dnAkt (lanes 4, 8) and treated with DMSO (lanes 1, 4, 5 & 8); LY294002 (lanes 2, 3, 6 & 7). e, Cytoplasmic or nuclear proteins from MCF-7 cells were immunoprecipitated with anti-p27kip1 and immunoblotted as indicated (lanes 1 & 2, respectively). Lane 3, 50 mug of protein lysate; lane 4, unrelated IgG were used as immunoprecipitation control. f, An immunocomplex kinase assay performed with endogenous Akt immunoprecipitated from MCF7 cells pretreated with DMSO (lanes 1−3) or LY294002 (lane 4) and stimulated with IGF-1 (lanes 1, 3 & 4). 6-His-tagged wild-type p27kip1 served as substrate. C, cytoplasmic proteins; N, nuclear proteins; Encircled P, phosphorylation. Immunoblots with antibodies to SP1 and gamma-tub were used as control of the purity and the integrity of nuclear and cytoplasmic protein extracts, respectively.



Full FigureFull Figure and legend (38K)
To determine whether Akt directly regulated the subcellular localization of p27kip1, MDA-MB468 and MCF-7 cells were transfected with empty vector or the constitutively active myristoylated hemagglutinine (HA)-tagged Akt (myrAkt) and trace amounts of farnesylated eukaryotic green fluorescent protein (pfEGFP) to track transfected and untransfected cells (Fig. 1a). After transfection, cells were divided into two plates, treated with DMSO or LY294002 for 24 hours and processed for flow cytometry. MyrAkt consistently relieved G1 arrest induced by LY294002 (Fig. 1a). Furthermore, myrAkt induced a marked decrease in the level of nuclear p27kip1 (Fig. 1d, lane 7) and a parallel reduction of the p27kip1 fraction complexed with nuclear CDK2, thereby increasing CDK2 kinase activity (by 70%) (Fig. 1d, lane 7). Transient transfection with a dominant-negative Akt mutant, in which the two activating phosphorylation sites (T308 and S473) were replaced by alanines (DN-Akt), caused p27kip1 to shift from the cytoplasmic to the nuclear compartment (Fig. 1d, lane 8).

We investigated whether direct phosphorylation by Akt promoted p27kip1cytoplasmic retention. p27kip1 was immunoprecipitated from fractionated cytoplasmic and nuclear lysates of MCF-7 cells and the immunocomplexes were blotted with anti-Akt or anti-p27kip1 (Fig. 1e). Akt-p27kip1 protein complexes were immunoprecipitated from breast cancer cells with anti-p27kip1 but not with control immunoglobulin G (IgG), and were restricted to the cytosolic compartment.

Finally, we determined whether endogenous Akt from MCF-7 breast cancer cells was able to phosphorylate p27kip1directly. Akt immunoprecipitates from MCF-7 cells that had been serum-starved, or stimulated with IGF-1 (100 ng/ml) in the presence or in the absence of LY294002 (20 muM), were used to phosphorylate 6-His-tagged recombinant p27kip1 in vitro (Fig. 1f). Anti-Akt, but not control IgG, immunoprecipitated a p27kip1-kinase from MCF-7 breast cancer cells, whose activity was stimulated by IGF-1 and abrogated by LY294002.

Akt phosphorylates T157 of p27kip1
Subsequently, we investigated whether phosphorylation of p27kip1 by Akt occurred in vivo. Analysis of the human p27kip1 sequence revealed a threonine residue, T157, within amino acids 152−157 (RKRPAT), which matches the minimal consensus for Akt-mediated phosphorylation (RxRxxT/S). T157 is located within the nuclear localization signal (residues 153−169) of p27kip1. To determine whether Akt phosphorylates threonine 157 of p27kip1, we performed an immunocomplex kinase assay. MyrAkt was transiently transfected into HEK293 cells. MyrAkt protein, recovered with antibodies to HA, phosphorylated 6-His-tagged recombinant p27kip1 protein in an immunocomplex kinase assay, whereas a kinase-dead form of Akt (Akt-K179M) did not (Fig. 2a). The same results were obtained with an active recombinant Akt kinase, whereas an inactive recombinant Akt was ineffective (Fig. 2a). The p27kip1 mutant, in which alanine substitutes for threonine at position 157 (p27kip1-T157A), was much less efficiently phosphorylated by myrAkt immunoprecipitates (about 25% residual phosphorylation) compared with the wild-type p27kip1 (Fig. 2a). This suggests that T157 is a major Akt phosphorylation site. We generated an affinity-purified polyclonal antibody that discriminates phosphorylated T157 from unphosphorylated T157 (anti-P-T157). Anti-P-T157 did not react with p27kip1-T157A, and reacted with 6-His-tagged p27kip1 only when it was preincubated with myrAkt. Reactivity was abrogated by treatment with calf intestinal phosphatase (CIP) (Fig. 2b).

Figure 2. Akt phosphorylates p27kip1 on threonine 157 in vitro and in vivo.
Figure 2 thumbnail

a, Immunocomplex kinase assays with recombinant active (lane 1) or inactive Akt (lane 2) or anti-HA immunoprecipitates of HEK293 cells transfected with myrAkt (lanes 4 & 6) or Akt-K179M (lanes 3 & 5); substrates were 6-His-tagged wild-type p27kip1 (lanes 1−4) or 6-His-tagged T157A mutant (lanes 5 & 6). b, Immunocomplex kinase assay with myrAkt immunoprecipitated from HEK293 cells (lanes 3−6). Substrates were wild-type (lanes 1, 3 & 5) or T157A p27kip1 (lanes 2, 4 & 6). In lanes 5 & 6, samples were pretreated with CIP. Phosphorylation of T157 was revealed by the anti-P-T157 antibody. c, FLAG-tagged wild-type p27kip1 (lanes 1, 2, 5 & 6) or T157A mutant (3 & 4) were transfected into HEK293 cells with myrAkt (lanes 2, 4 & 6) or Akt-K179M (lane 5). Cells were labeled with 32P-ortho-phosphate, total proteins were immunoprecipitated with anti-FLAG (lanes 1−5) or with control IgG (lane 6) and autoradiographed. d, FLAG-tagged wild-type p27kip1 (lanes 1, 2 & 5) or T157A mutant (3 & 4) were transfected into HEK293 cells with myrAkt (lanes 2 & 4) and immunoblotted with anti-P-T157. In lane 5 the sample was pretreated with CIP.



Full FigureFull Figure and legend (17K)
Subsequently, we investigated whether phosphorylation of p27kip1 depends on T157. FLAG-tagged wild-type and mutant p27kip1 (FLAG-p27kip1 and FLAG-p27kip1-T157A) were transiently transfected in HEK293 cells together with an excess of myrAkt, Akt-K179M or empty vector. Cells were metabolically labeled with 32P-ortho-phosphate and collected; p27kip1 was recovered with anti-FLAG. p27kip1 phosphorylation was greatly enhanced when coexpressed with myrAkt but not with the kinase-dead Akt. MyrAkt-induced phosphorylation of the p27kip1-T157A mutant was significantly lower (40% residual) compared with the wild-type p27kip1 ( Fig. 2c). Similar results were obtained when p27kip1 phosphorylation was revealed by anti-P-T157. Again, the specificity of anti-P-T157 was demonstrated by the lack of reactivity upon treatment of lysates with CIP (Fig. 2d).

Akt-mediated relocalization of p27kip1 depends on T157
HEK293 cells were transfected with FLAG-p27kip1 and FLAG-T157A-p27kip1 with and without myrAkt, treated with LY294002 to suppress background endogenous PI3K activity, and either processed for indirect immunofluorescence or fractionated to enrich for nuclear or cytoplasmic proteins. Fig. 3a shows a representative example of immunofluorescence. With LY294002, p27kip1 accumulated in the nucleus (>95% of cells); with myrAkt, it accumulated in the cytoplasm (>60% of cells). Unlike the wild-type protein, p27kip1-T157A localized almost exclusively (>80%) in the nucleus even when coexpressed with myrAkt (Fig. 3b). Western blotting of cytoplasmic and nuclear proteins confirmed these results (Fig. 3c).

Figure 3. Akt-dependent phosphorylation of T157 promotes cytosolic localization of p27kip1 and rescues S-phase entry of p27kip1-transfected cells.
Figure 3 thumbnail

a, myrAkt (10 mug) or empty vector was cotransfected with FLAG-tagged wild-type (wt) p27kip1 or its T157A mutant (1 mug) in HEK293 cells, plated onto coverslips and treated with LY294002. The cellular localization of p27kip1 was detected with anti-FLAG. Red arrows indicate nuclear p27kip1; yellow arrows indicate cytoplasmic p27kip1. b, Bars represent average of 4 experiments in which at least 500 transfected cells were counted. , nuclear proteins; , cytoplasmic proteins. Error bars indicate s.d. c, Immunoblot analysis of p27kip1 localization in fractionated HEK293 cells transfected with wild-type (lanes 1, 3, 5 & 7) or T157A mutant p27kip1 type (lanes 2, 4, 6 & 8) in the presence of control vector (lanes 1, 2, 5 & 6) or myrAkt (lanes 3, 4, 7 & 8). d, BrdU incorporation in HEK293 cells cotransfected with FLAG-tagged wild-type or T157A p27kip1 (1 mug) and myrAkt or the empty vector (10 mug), plated onto coverslips and treated with LY294002. p27kip1-transfected cells were identified with a polyclonal anti-p27kip1 antibody, whereas cells incorporating BrdU were identified with monoclonal anti-BrdU. e, Immunoblot analysis of the expression of FLAG-tagged wild-type (lanes 1 & 2) or T157A p27kip1 (lanes 3 & 4) and myrAkt (lanes 2 & 4) constructs in HEK293 cells.



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To investigate the effects exerted by myrAkt on p27kip1-mediated cell cycle arrest, HEK293 cells were transfected with wild-type p27kip1 or the T157A mutant and an excess of myrAkt or the empty vector, and the rate of BrdU incorporation in p27kip1-positive transfected cells was calculated as a measure of S phase entry. As shown in Fig. 3d, the expression of p27kip1 or p27kip1-T157A blocked BrdU incorporation in all transfected cells (<1% of p27kip1-positive cells incorporated BrdU). MyrAkt consistently relieved the block imposed by wild-type p27kip1, as evidenced by the 11-fold increase in Brdu incorporation in myrAkt-transfected cells. Notably, the p27kip1-T157A mutant was resistant to myrAkt: BrdU incorporation of p27kip1-T157A being increased only about two-fold by myrAkt. In contrast, the growth-arresting effects of the unrelated p27kip1-T187A mutant remained sensitive to myrAkt inhibition (data not shown). To demonstrate equal expression levels, we performed western-blot analysis of HEK293 cells transfected with FLAG-tagged p27kip1 and T157A-p27kip1 mutants with or without myrAkt (Fig. 3e).

The PI3K-Akt pathway regulates the accumulation of T157-phosphorylated p27kip1 in the cytoplasmic compartment of breast cancer cells. Using the specific anti-P-T157 antibody, we found p27kip1 phosphorylated at T157 in proliferating MDA-MB468 breast cancer cells; exposure for 24 hours to LY294002 inhibited Akt and strongly reduced T157 phosphorylation (Fig. 4a, lane 2). Notably, phosphorylation of endogenous p27kip1 at T157 in proliferating cells was restricted almost exclusively to the cytoplasmic compartment. Conversely, treatment of MDA-MB468 cells with PD98059 (50 muM) or U0126 (5 muM) had little effect on the phosphorylation of p27kip1 at T157 (Fig. 4a, lane 3 and data not shown). Stable clones of MDA-MB468 (3 independent clones) transfected with myrAkt (MDA-MB468-myrAkt) showed increased levels of p27kip1 phosphorylation at T157 and cytosolic accumulation even in the presence of LY294002 or PD98059 (Fig. 4a, lanes 7−9). To determine the extent of p27kip1 phosphorylation, cytosolic extracts from MDA-MB468-myrAkt cells were immunodepleted of T157-phosphorylated p27kip1 by three rounds of immunoprecipitation with anti-P-T157-p27kip1 or antibodies against phosphorylated Akt substrates. The fraction of phosphorylated p27kip1 in MDA-MB468-myrAkt cells was assessed by Western blot performed with anti-p27kip1 on equal amounts of the original and depleted extracts (Fig. 4c). The fraction of phosphorylated p27kip1 in MDA-MB468-myrAkt cells was about 40−60%, as determined by PhosphorImager quantification (Fig. 4c, lanes 1 & 2). Finally, as in HEK293 cells, in phosphorylation of p27kip1 at T157 is required for the regulation of p27kip1 compartmentalization MDA-MB468 cells. In fact, transfected wild-type FLAG-tagged p27kip1 localized mainly to the cytosolic compartment, but accumulated in the cell nucleus upon exposure to LY294002. By contrast, p27kip1-T157A was localized in the cell nucleus independently of PI3K-Akt activity (Fig. 4d).

Figure 4. In breast cancer cells the PI3K-Akt pathway promotes T157 phosphorylation and cytosolic retention of p27kip1.
Figure 4 thumbnail

a, MDA-MB468 and MDA-MB468-myrAkt were treated with DMSO (lanes 1 & 4 and 7 & 10, respectively), LY294002 (lanes 2 & 5 and 8 & 11, respectively) or PD89059 (lanes 3 & 6 and 9 & 12, respectively) and fractionated to determine by immunoblot localization and phosphorylation status of endogenous p27kip1. b, Western-blot analysis of the effects of PD89059 and LY294002 on the activity and the expression of Erk1/2 and Akt in MDA-MB468 and MDA-MB468-myrAkt cells. Treatments were as follows: DMSO (lanes 1 & 4); LY294002 (lanes 2 & 5); PD98059 (lanes 3 & 6). c, Cytosolic extracts of MDA-MB468-myrAkt cells immunodepleted of phosphorylated p27kip1 by immunoprecipitation with antibodies against phosphorylated Akt substrates (upper panel) or anti-P-T157 p27kip1 (lower panel) and were analyzed by immunoblot with a pan-p27kip1 antibody. d, MDA-MB468 cells were transfected with 10 mug of FLAG-p27kip1 (lanes 1, 2, 5 & 6) or FLAG-p27kip1-T157A mutant (lanes 3, 4, 7 & 8). Transfected cells were divided into 2 dishes, treated with DMSO or LY294002, and fractionated to determine the phosphorylation status of T157 and the subcellular localization of exogenous p27kip1.



Full FigureFull Figure and legend (40K)
Akt phosphorylates p27kip1 in normal cells
We analyzed the effects of Akt activation in three different types of normal cells: an immortalized non-tumorigenic cell line from normal mammary epithelium (MCF-10A), a primary culture of normal human mammary epithelial cells (HMECs) and a primary culture of normal human lung fibroblasts (IMR-90). myrAkt (10 mug) or the empty vector was cotransfected with FLAG-tagged wild-type p27kip1 or its T157A mutant (1 mug) in MCF-10A, HMEC or IMR-90 cells, plated onto coverslips and treated with LY294002. The cellular localization of p27kip1 was detected with anti-FLAG antibodies. In all three cellular systems, myrAkt induced cytoplasmic accumulation of wild-type p27kip1 but not of the T157A mutant (Fig. 5a and b, and data not shown). Moreover, the pharmacological inhibition of the PI3K-Akt pathway by LY294002 in MCF-10A and HMEC cells impaired p27kip1 phosphorylation at T157 (Fig. 5c and d, lane 2), caused p27kip1 nuclear accumulation (Fig. 5c and d, lane 5) and inhibited cell growth (data not shown). Conversely, inhibition of the MEK-Erk pathway in MCF-10A and HMECs by PD98059 had little effect on p27kip1 phosphorylation at T157 (Fig. 5c and d, lane 3) and p27kip1 nuclear accumulation (Fig. 5c and d, lanes 4−6). Notably, the fraction of p27kip1 phosphorylated at T157 in untransfected HMECs as determined by immunodepletion experiments was 15−25% (data not shown). These results suggest that Akt plays an essential role in modulating p27kip1 localization in normal human breast cells, and that an increase in the level of phosphorylated p27kip1 may contribute to the impairment of p27kip1 function in human breast cancer.

Figure 5. The PI3K-Akt pathway regulates T157 phosphorylation and nuclear accumulation of p27kip1 in normal mammary cells.
Figure 5 thumbnail

a and b, myrAkt (10 mug) or the empty vector was cotransfected with FLAG-tagged wild-type p27kip1 or its T157A mutant (1 mug) in MCF-10A (a) and HMEC (b) cells, plated onto coverslips and treated with LY294002. The cellular localization of p27kip1 was assessed with anti-FLAG. Red arrows indicate nuclear p27kip1; yellow arrows indicate myrAkt and cytoplasmic p27kip1. c, Immunoblot analysis of PI3K- or MEK-Erk-dependent p27kip1 subcellular localization in MCF-10A cells. Cells were treated with DMSO (lanes 1 & 4); LY294002 (lanes 2 & 5); PD98059 (lanes 3 & 6). Right panels, cells were treated with DMSO (lane 1), LY294002 (lane 2) or PD98059 (lane 3). d, Immunoblot analysis of PI3K- or MEK-Erk-dependent p27kip1 subcellular localization in HMEC cells. Left panels, cells were treated with DMSO (lanes 1 &4), LY294002 (lanes 2 & 5) or PD98059 (lanes 3 & 6). Right panels, cells were treated with DMSO (lane 1), LY294002 (lane 2) or PD98059 (lane 3).



Full FigureFull Figure and legend (77K)
Akt activation and p27kip1 localization in breast cancer
We determined p27kip1 protein expression by immunoblot in 54 human primary breast carcinomas and 3 samples of normal mammary tissue. p27kip1 was absent from about 26% of breast tumors (14 of 54). We evaluated the extent of Akt phosphorylation in the 40 p27kip-positive breast cancer specimens by immunoblot with the phospho-specific anti-S473 Akt and measured the pAkt:Akt ratio by Phosphorimager scanning. Samples segregated into 15 tumors with a low (<0.1) pAkt:Akt ratio (group 1), 10 tumors with a pAkt:Akt ratio between 0.1 and 0.8 (group 2) and 15 tumors with a pAkt:Akt ratio >0.8 (group 3). We determined the subcellular localization of p27kip1 in the p27kip1-positive samples using immunostaining with anti-p27kip1. As described8, p27kip1 was scored 'nuclear' when more than 50% of p27kip1-expressing cells presented nuclear staining, and 'cytoplasmic' when there was nucleo-cytoplasmic or exclusively cytoplasmic staining in at least 35% of p27kip1-expressing cells. At least 500 cells in five independent fields were counted at high-field magnification (times100) for each tumor (Fig. 6b). According to these criteria, p27kip1 protein was detected in the nuclei of normal breast epithelium (data not shown) and in the nucleus and cytoplasm or cytoplasm in 15 of 40 breast tumors (37.5%). The subcellular localization of p27kip1 was also determined by immunoblot in 19 of 40 primary breast tumors for which lysates enriched in cytoplasmic and nuclear proteins were available. The results of protein fractionation were consistent with the localization of p27kip1 determined by immunostaining (Fig. 6c).

Figure 6. Activation of Akt correlates with T157 phosphorylation and cytosolic accumulation of p27kip1 in breast cancer.
Figure 6 thumbnail

a, Akt activation and p27kip1 T157 phosphorylation in primary breast cancer tissues from patients. Ponceau S staining ensured uniform loading (data not shown). b, p27kip1 localization in primary breast cancer by immunostaining: upper panel, a representative case with nuclear p27kip1 staining; lower panel, a representative tumor with cytoplasmic p27kip1. c, Analysis of Akt phosphorylation, p27kip1 T157 phosphorylation and p27kip1 localization in primary breast cancer tissues from patients by immunoblot on fractioned lysates and p27kip1 immunoprecipitates. d, Correlation between Akt activation and p27kip1 phosphorylation at T157 (blue bars) and p27kip1 sub-cellular localization (red bars). Group 1, tumors without Akt S473 phosphorylation (n = 15); group 2, tumors with low-to-medium Akt S473 phosphorylation (n = 10); group 3, tumors with high Akt S473 phosphorylation (n = 15).



Full FigureFull Figure and legend (47K)
Subsequently, we assessed T157 phosphorylation of p27kip1 in human breast cancers using anti-P-T157 immunoblotting of p27kip1 immunoprecipitates. T157-phosphorylated p27kip1 was detected in 13 of 40 cases (32.5 %). Notably, most tumors that were positive for T157-phosphorylated p27kip1 presented p27kip1 in the cytoplasmic compartment, whereas the remaining cases, negative to the antibody to P-T157, had nuclear staining. T157-phosphorylated p27kip1 was almost exclusively confined to the cytosol (Fig. 6c). T157 phosphorylation of p27kip1 (Fig. 6d, red bars) occurred in 40% of group 2 tumors and in 60% of group 3 tumors, but in only 6% of group 1 tumors. The correlation between Akt activation and T157 phosphorylation of p27kip1 was statistically significant (P < 0.0001). Cytoplasmic localization of p27kip1 (red bars) occurred in 40% of group 2 tumors, 67% of group 3 tumors and in no group-1 tumor (Fig. 6c) (P < 0.0001). The correlation between Akt activation and localization of p27kip1 was statistically significant (P < 0.0001; chi2 test).

Discussion
Hyperactivation of the PI3K-Akt pathway is critical in human tumorigenesis because it promotes cell growth, survival and resistance to treatment24, 36. The high frequency of breast carcinomas in patients with Cowden's syndrome in which Akt is activated by loss of PTEN (ref. 31), and the poor prognosis of breast cancer patients associated with Akt activation, implicated deregulation of PI3K-Akt pathway in breast tumorigenesis37. Akt disrupts normal cell-cycle regulation by affecting the expression of cyclin D and p27kip1 and the localization of cyclin D (refs. 38,39) and p21cip1 (ref. 29). Transcriptional down-regulation of p27kip1 mRNA by Akt occurs through relocalization and subsequent inhibition of the forkhead transcription factor (AFX). However, downregulation of p27kip1 mRNA is not frequently observed in human cancer. Conversely, Akt-dependent stabilization of cyclin D1, mediated by phosphorylation-dependent GSK3 inhibition38, 39, may play a role in the overexpression of cyclin D1 observed in breast carcinomas.

Thus far, the only regulator of cell cycle progression that has been demonstrated to be phosphorylated by Akt is p21cip1. In fact, p21cip1 relocalizes to the cytoplasmic compartment in breast cancer cells upon Akt phosphorylation. However, the significance of p21cip1 phosphorylation in the development of human cancer remains obscure as p21cip1 mislocalization has not been reported as a frequent event.

Here we describe a novel mechanism whereby p27kip1 is functionally disrupted in breast cancer. We demonstrate that Akt phosphorylates p27kip1 in vitro at threonine 157, as well as in living cells using metabolic labeling and a phospho-specific antibody that discriminates the phosphorylated form of p27kip1. Several lines of evidence demonstrate that Akt-dependent T157 phosphorylation is necessary to promote cytoplasmic accumulation of p27kip1. In proliferating breast cancer cells, most p27kip1 is localized in the cytosolic compartment. Pharmacological inhibition of PI3K or expression of a dominantly interfering Akt construct induced nuclear accumulation of p27kip1, whereas constitutively active Akt induced cytoplasmic accumulation of p27kip1. In contrast, neither LY294002 nor expression of myrAkt affected the nuclear localization of a p27kip1-T157A mutant. These effects were specific to P13K-Akt as they were not observed when the MEK-Erk signaling pathway was inhibited. The effects of Akt on p27kip1 phosphorylation and localization were observed also in primary mammary cells, which suggests that this mechanism may operate in normal cells.

In breast cancer cells, p27kip1 phosphorylation at T157 was modulated by Akt activity and occurred almost exclusively in the cytoplasmic compartment. This probably reflects impaired nuclear import of p27kip1 (J. Slingerland, pers. comm.). Additional mechanisms could be the binding of phosphorylated p27kip1 to cytoplasmic anchors such as 14-3-3 proteins, as indicated by our preliminary results (unpublished data) or cyclin D complexes as suggested by previous studies8, 40.

Akt-mediated cytosolic accumulation of p27kip1 is critical for Akt mitogenic signaling. In fact, when forced to localize to the cytoplasm, p27kip1 is less efficient in inhibiting growth8. Furthermore, Akt-mediated exclusion of wild-type p27kip1 from the nuclear compartment results in activation of nuclear CDK2 and cell-cycle progression, whereas mutation in T157 confers resistance to Akt-mediated p27kip1 nuclear exclusion and impairs Akt-dependent rescue of p27kip1-induced cell-cycle arrest.

However, it is necessary to point out that, in addition to T157, our results suggest the existence in p27kip1 of secondary Akt-dependent phosphorylation sites, which accounts for 25−40% of Akt-mediated phosphorylation of p27kip1. This is intriguing because T157 is not conserved in all mammals, it is present in monkeys and felines but not in rodents. Notably, species-restricted phosphorylation of p27kip1 by Akt is reminiscent of Akt phosphorylation of caspase 9 (ref. 26). These observations raise the possibility that in different species, Akt-dependent phosphorylation at different sites may contribute to the regulation of p27kip1 localization. Finally, although our results are suggestive of Akt phosphorylation of p27kip1, we cannot exclude that other kinases may phosphorylate p27kip1 at T157 and thus regulate the sub-cellular localization of p27kip1 independently of Akt.

Cytoplasmic displacement of p27kip1 occurs in various human tumors (such as thyroid, oesophageal and colorectal carcinomas and sarcomas)8, 16, 17. However, the pathways responsible for altered p27kip1 compartmentalization in cancer cells are not known. We propose a model whereby the constitutive hyperactivation of Akt promotes cancer development (at least in part) by inducing phosphorylation of p27kip1 at T157 and, as a consequence, cytoplasmic retention of p27kip1 and impairment of its growth-inhibitory properties.

In breast cancer, loss of p27kip1 expression occurs through increased protein turnover13. However, in a significant fraction of tumors, the p27kip1 protein level is not significantly downregulated13. Here, in 37.5% of p27kip1-positive breast carcinomas, p27kip1 was located in the cytoplasmic or nucleo-cytoplasmic compartment. Notably, T157-phosphorylation of p27kip1, detected mainly in the cytoplasmic compartment of tumor cells, occurred in most cancers with cytoplasmic p27kip1. This finding points to a causal link between p27kip1 phosphorylation at T157 and subcellular localization. Furthermore, tumors in which p27kip1 was phosphorylated at T157 and localized in the cytoplasmic compartment had high levels of activated Akt, whereas in tumors without T157 phosphorylation of p27kip1 the protein was in the nucleus and Akt inactive. In conclusion, our study identifies a novel mechanism whereby the CDK inhibitor p27kip1 is functionally inactivated in cancer and demonstrates that Akt-dependent regulation of p27kip1 localization may be critical in the development of human breast cancer.

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Methods
Cell lines.
The HEK293, IMR-90, MCF-10, MCF7, MDA-MB231 and MDA-MB468 human cell lines were from the American Tissue Cell Culture collection. HMECs were from Clonetics (BioWhittaker, Walkersville, Md). Cells were grown in DMEM (Life Technology, Paisley, Pennsylvania) containing 10% FCS (Life Technology) except for HMEC that were grown according to manufacturer instructions. PD98059, LY290042, U0126, IGF-1 and Okadaic Acid were from Sigma (Sigma, St. Louis, Missouri).

Constructs and transfections.
Akt constructs have been described41. The wild-type p27kip1 construct has been described42, 43. The p27kip1-T187A and p27kip1-T157A mutants were generated with a site-specific mutagenesis kit (Stratagene, La Jolla, California) and confirmed by DNA sequencing. Cells were transfected with Fugene 6 (Roche, Basel, Switzerland). The average transfection efficiency of HEK293 cells was about 80−90%. The average transfection efficiency of breast cancer cells was about 40%.

BrdU incorporation and indirect immunofluorescence.
Cells were grown on coverslips, transfected and labeled 48 h post-transfection with BrdU at a concentration of 10 muM, fixed in 3% paraformaldehyde and permeabilized with 0.2% Triton X-100. p27kip1 and BrdU-positive cells were recognized with fluorescein (FITC)- or Texas-Red-conjugated secondary antibodies, respectively (Jackson ImmunoResearch Laboratories, Philadelphia, Pennsylvania)42, 43. Cell nuclei were identified by Hoechst staining. Fluorescence was visualized with a Zeiss 140 epifluorescent microscope equipped with filters that discriminated between Texas-Red and fluorescein. The results are the average of 4 experiments plusminus s.d. in which at least 500 transfected cells were counted. Error bars indicate s.d.

Flow-cytometric analysis.
Breast cancer cells were analyzed for DNA content as described42. DNA was stained with propidium iodide (50 mug/ml) and analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, California) interfaced with a Hewlett Packard computer (Palo Alto, California). Cells were gated to isolate fEGFP-transfected cells and the cell cycle distribution was analyzed with the CELL-FIT program (Becton Dickinson).

Protein extraction, western blotting and antibodies.
The antibodies used in this study were: anti-FLAG (M5; Sigma); anti-CDK2 (M2) (Santa Cruz Biotechnology, Santa Cruz, California); anti-p27kip1 (Transduction Laboratories, Lexington, Kentucky); anti-Akt and anti-phosphoAkt (Ser473) (New England Biolabs, Lake Placid, New York); phospho-(Ser/Thr) Akt substrate antibody, raised against the Akt consensus phosphorylation sequence (RXRXXT/S) (#9619, Cell Signaling Technology, Beverly, Massachusetts). Anti-P-T157 was purchased from Neosystem (Strasbourg, France). The antibody was obtained by immunizing rabbits against the pepide YRKRPApTDDSSTQNKR and purified through passage on a column linked to the unphosphorylated peptide followed by a final purification on a column linked to the phosphorylated peptide. Nuclear or cytoplasmic proteins were differentially extracted by lysing cells in ice-cold hypotonic buffer (0.2% NP-40, 10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl). Nuclei were separated through a 30% sucrose cushion and lysed in hypertonic buffer (250 mM Tris-HCl, pH 7.8, 60 mM HCl). Antibodies to SP1 and gamma-tubulin were used to assess the purity of the fractions. Immunoblots were performed as described8, 42, 43.

Immunodepletion of phosphorylated T157 p27kip1.
Cytosolic extracts from MDA-MB468-myrAkt cells (350 mug) were depleted of p27kip1 phosphorylated at T157 by 3 rounds of immunoprecipitation with antibodies to P-T157-p27kip1 or to phosphorylated Akt substrates. Equal amounts (20 mug) of the immunodepleted lysate and of the original extract were fractionated by SDS−PAGE and analyzed by immunoblot with a pan-p27kip1 antibody.

Preparation of recombinant p27kip1 and in vitro kinase assays.
The cDNA encoding human p27kip1 was cloned into the pET21a vector (Novagen, Madison, Wisconsin) in-frame with the 6-His tag at the C terminus. Recombinant p27kip1 was purified using Ni-NTA resin (Qiagen GmbH, Germany) as described44. Akt immune complexes were prepared by immunoprecipitation with anti-HA antibodies (Santa Cruz Biotechnology) and collected on protein A-Sepharose beads and incubated as described25. Recombinant Akt was from Upstate Biotechnology (Lake Placid, New York). The phosphorylated substrates were separated on 12.5% SDS−PAGE and quantified by PhosphorImager (GS525, Biorad, Hercules, California).

In vivo labeling of cells.
Transiently transfected HEK293 cells were incubated overnight in phosphate-free medium supplemented with 10% dialyzed FBS and subsequently metabolically labeled with 1 mCi/ml 32P-ortho-phosphate (Amersham Pharmacia Biotech, Little Chalfont, UK) for 4 h at 37 °C. Proteins were immunoprecipitated with specific antibodies or with preimmune mouse or rabbit serum (NMS or NRS), fractionated onto polyacrylamide SDS gel, transferred to nitrocellulose filters and subjected to autoradiography. The incorporated radioactive phosphate was determined by PhosphorImager analysis and normalized to the abundance of p27kip1 in the immunoprecipitates.

Tissue samples and immunoperoxidase staining of paraffin-embedded tissues.
Tumor samples were obtained from patients who underwent surgery at the National Cancer Institute, Naples, Italy. Immunohistochemistry was performed with monoclonal anti-p27kip1 antibodies (k25020, Transduction Laboratories) as previously described8. Antigens were retrieved by microwave irradiation. Statistical analysis was performed by use of the chi2 test using the Graphpad Prism software (Graphpad, San Diego, California) (P < 0.0001). Animal and Human protocols included in this study were approved by the Scientific board of the Instituto di Endocrinologia Sperimentale del CNR.

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Received 8 April 2002; Accepted 14 August 2002; Published online: 16 September 2002.

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