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Cancer Research 67, 8032, September 1, 2007. doi: 10.1158/0008-5472.CAN-07-0083
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Genetic Screening Reveals an Essential Role of p27kip1 in Restriction of Breast Cancer Progression

Yuhui Yuan1, Li Qin1, Dan Liu2, Ray-Chang Wu1, Paola Mussi1, Suoling Zhou1, Zhou Songyang2 and Jianming Xu1

Departments of 1 Molecular and Cellular Biology and 2 Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas

Requests for reprints: Jianming Xu, Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Phone: 713-798-6199; Fax: 1-713-798-3017; E-mail: jxu{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The genetic changes and mechanisms underlying the progression of estrogen-dependent breast cancers to estrogen-independent, antiestrogen-resistant, and metastatic breast cancers are unclear despite being a major problem in endocrine therapy. To identify genes responsible for this progression, we carried out a genetic screening by an enhanced retroviral mutagen (ERM)–mediated random mutagenesis in the estrogen-dependent T47D breast cancer cells. We found that T47D cells contain only one p27kip1 (p27) allele coding for the p27 cyclin-dependent kinase (CDK) inhibitor. An ERM insertion into the p27 locus of T47D cells disrupted the p27 gene and created estrogen-independent and antiestrogen-resistant breast cancer cells that still maintained functional estrogen receptors. Disruption of p27 in T47D cells resulted in several changes, and most of these changes could be rescued by p27 restoration. First, CDK2 activity was increased in the absence of estrogen or in the presence of estrogen antagonists tamoxifen or ICI 182780; second, amplified in breast cancer 1 (AIB1), a cancer overexpressed transcriptional coactivator, was hyperphosphorylated, which made AIB1 a better coactivator for E2F1; and third, growth factor receptor binding protein 2–associated binder 2 (Gab2) and Akt activity were increased following E2F1 overactivation, leading to a significant enhancement of cell migration and invasion. Furthermore, the p27-deficient cells, but not T47D control cells, developed lung metastasis in an ovarian hormone–independent manner when they were i.v. injected into nude mice. In sum, loss of p27 activated AIB1, E2F1, Gab2, and Akt; increased cell migration and invasion; caused antiestrogen insensitivity; and promoted metastasis of breast cancer cells. These findings suggest that p27 plays an essential role in restriction of breast cancer progression. [Cancer Res 2007;67(17):8032–42]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In estrogen-dependent breast cancer cells, estrogen, through estrogen receptor {alpha} (ER{alpha}), enhances c-Myc and cyclin D1 expression, down-regulates p27, and activates cyclin E/cyclin-dependent kinase (CDK)-2 to promote G1-S transition (1, 2). Because most primary breast cancers express ER{alpha} and require estrogen to grow, estrogen antagonists and aromatase inhibitors such as tamoxifen and letrozole are used to treat estrogen-dependent breast cancers (3–5). Although these treatments are initially effective, acquired resistances are a major problem. In most cases, development of drug resistance is not due to a loss or mutation of ER{alpha} (4, 6).

Overexpression or activation of receptor tyrosine kinases (RTK) and Ras oncoproteins is common in cancers. Human epidermal growth factor receptor 2 (HER2)/neu overexpression happens in 20% to 30% breast cancers and correlates with more aggressive cancer phenotypes and tamoxifen resistance (7, 8). Ras and RTKs including HER2, insulin-like growth factor I receptor, and epidermal growth factor receptor activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (9–12). This pathway plays a pivotal role in cell survival, proliferation, motility, tumorigenesis, and metastasis through phosphorylation and subsequent relocalization of key regulatory molecules such as p27 (13–15).

In cell nucleus, p27 associates with cyclin E-CDK2 and inhibits Rb hyperphosphorylation to keep cells in G1 phase (16). Mitogenic stimuli cause p27 phosphorylation, ubiquitination, degradation, and translocation to the cytoplasm and increase cyclin E-CDK2 activity, leading to G1-S transition through Rb phosphorylation and E2F activation (16). In breast cancers overexpressing HER2, Akt phosphorylates p27 and keeps p27 in the cytoplasm, which precludes p27-induced G1 arrest (13–15). Therefore, both total p27 reduction and p27 exclusion from the nucleus of breast cancer cells are associated with poor prognosis and estrogen independence (16, 17). Down-regulation of p27 also enhances MCF-7 breast cancer cell growth in the presence of antiestrogens (18, 19). However, despite many studies correlating p27 levels and locations with tumorigenesis, the lack of p27 null mutation in human tumors has made it difficult to understand the exact role of p27 in breast cancer progression (20). Furthermore, the fact that oncogene-induced mammary tumorigenesis is accelerated in p27+/– mice but suppressed in p27–/– mice suggests a complex role of p27 in breast cancer (21).

E2F is the key transcriptional factor for cell cycle progression (16). E2F1 interacts with amplified in breast cancer 1 (AIB1), and this interaction potentiates E2F1 target gene expression (22, 23). AIB1 is a transcriptional coactivator for nuclear receptors and other transcription factors including E2F1 (22, 24). AIB1 is overexpressed in ~60% of human breast tumors (25). Overexpression of AIB1 in mouse mammary epithelium causes mammary carcinomas (26), whereas inactivation of AIB1 in mice suppresses oncogene and carcinogen-induced mammary tumorigenesis (27, 28). These findings indicate that AIB1 is an oncogene. AIB1 activity is not only determined by its concentration but also regulated by phosphorylation (29). In addition, E2F1, through up-regulation of the adaptor protein growth factor receptor binding protein 2–associated binder 2 (Gab2), strongly activates Akt (30), which promotes cell motility, invasion, and cancer metastasis (31, 32). Thus, we hypothesize that the serial events initiated by p27 inactivation may promote breast cancer cell migration, invasion, and metastasis.

In this study, we have done a genetic screening by using the enhanced retroviral mutagen (ERM) system (33) and identified the p27 loss-of-function clone. We show that p27 deficiency in T47D cells causes estrogen-independent and antiestrogen-resistant growth; stimulates AIB1 phosphorylation and its coactivator activity for E2F1; promotes Akt activity, cell motility, and invasion; and results in lung metastasis in ovariectomized nude mice injected i.v. with these cells.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Retroviral infection. T47D cells (104) were infected with MSCV-Eco-zeo and MSCV-SV-tTA viruses (multiplicity of infection, 10:1) and selected in medium with 200 µg/mL G418 (33). The surviving T47DtTA cells were expanded to 107, transferred into six-well plates (5 x 105 per well), and spin-infected with ERM retroviruses. Each of the three ERM retroviral vectors carried an ERM promoter, an expression tag in one of the three reading frames, and a splice donor (Fig. 1B ; ref. 33). Two days after ERM infection, cells were transferred to 100-mm dishes for a 30-day selection in RPMI 1640 containing 10 µg/mL insulin, 10% charcoal dextran–stripped FCS (CSFCS), and 1 µg/mL puromycin. For p27 expression, the COOH-terminally poly-histidine–tagged p27 cDNA was taken from the pCMV-p27 plasmid (a kind gift from Dr. Mong-Hong Lee, Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX) and subcloned into the pQCXIH retroviral plasmid (BD Biosciences). The pQCXIH-p27 retroviruses were produced in PT67 packaging cells.


Figure 1
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  		Figure 1. Disruption of p27 in T47D cells causes loss of G1-S checkpoint and estrogen-independent growth. A, T47DtTA (tTA) and T47DtTA/p27m (p27m) cells were cultured in medium containing BrdUrd and 10% FCS or 10% CSFCS. BrdUrd incorporation and DNA content per cell were analyzed by fluorescence labeling and flow cytometry. Cell fractions (%) are indicated. Note the increase in G2-M fraction of T47DtTA/p27m cells cultured in medium with FCS and the unchanged proliferation status of T47DtTA/p27m cells cultured in medium with CSFCS. B, the p27 gene structure and the insertional disruption of p27 by ERM. The first and second exons of p27 and p27 protein are outlined. The ERM insertion site and the locations of four primers (P1–P4) used for PCR are indicated. White box, noncoding regions; gray box, amino acid coding regions; SD, splice donor; stretched box, the ERM tag. CBD, cyclin binding domain; CDKBD, cyclin dependent kinase binding domain; NLS, nuclear localization signal. C, PCR analysis of the p27 gene. Note the absence of the 985-bp P1/P3 product of the wild-type allele and the presence of 523-bp P4/P3 product of the ERM allele in T47DtTA/p27m cells. The 474-bp P1/P2 products were amplified from exon 1 and served as a positive control for the PCR. The ERM insertion was longer than 3 kb and thereby PCR using P1/P3 and Taq DNA polymerase was unable to amplify this long fragment in T47DtTA/p27m cells. D, immunoblotting analyses with antibodies against p27 COOH terminus ({alpha}-C; BD Transduction Lab) and p27 NH2 terminus ({alpha}-N; Santa Cruz Biotechnology). Note the absence of full-length p27 (lanes 1 and 4) and the presence of p27-C40 (lane 2) and p27-N158 (lane 4) in T47DtTA/p27m cells.

 
Reverse transcription-PCR for identification of ERM-targeted genes. Colonies were isolated and cultured in RPMI 1640 with 10 µg/mL insulin and 10% CSFCS. RNA was prepared and reverse transcribed with primer 5'-GCAAATACGACTCACTATAGGGATCCNNNN(G/C)ACG. PCR was done using ERM-tag primer (5'-ACCATGGGGAGCAGCAAGAGCAAACCAAAAGACCCCAGCCAACGC) and T7 primer. PCR products were sequenced and GenBank was searched to identify ERM-targeted genes.

Cell cycle analysis, colony formation assay, immunochemical analyses, kinase assay, transfection assay, and migration and invasion assays. These assays were carried out by following standard methods as previously described (28, 29, 34). For details, see Supplementary Materials and Methods.

Tumor growth and experimental metastasis assays. Mouse protocols were approved by Baylor College of Medicine Animal Care and Use and Committee. For tumor growth in mammary glands, 8 x 106 cells were mixed with Matrigel and injected into both fourth mammary fat pads of ovariectomized athymic nude mice (35). Each type of cells was injected into six mice without pellets and six mice with 60-day estradiol-releasing pellets (1.7 mg/pellet from Innovative Research). Four weeks after injection, tumors were excised for histology and immunohistochemistry. Other organs including lung, liver, and mesenteric lymph nodes were examined for metastasis. For experimental metastasis, 106 cells were injected into the tail lateral vein of ovariectomized nude mice. Three weeks after injection, their lungs were examined for metastasis and processed for histology and immunohistochemistry as described (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of estrogen-independent growth mutants. T47D cells express ER{alpha} and depend on estrogen to grow and survive. A genome-wide screening using ERM was used to identify genes with gain-of-function or loss-of-function mutations that could make T47D cells survive in estrogen-free medium. To prepare cells for ERM infection, T47D cells were first infected with MSCV-Eco-zeo viruses to express the mouse ecotropic receptor and with MSCV-SV-tTA to express tTA tetracycline-off regulator. The obtained stable T47DtTA cells were subsequently infected with ERM and growth selected in estrogen-free medium. As expected, no colonies were found from uninfected T47DtTA cells after culturing in the estrogen-free medium. In contrast, ~150 colonies formed from a total of 5 million ERM-infected T47DtTA cells in the selection medium. Surviving colonies were isolated and the ERM-targeted genes were identified in each clone with the use of reverse transcription-PCR (RT-PCR) that amplified ERM expression tag and its trapped 3' exon sequences (33). Of 15 clones analyzed, we obtained 12 RNA sequences containing ERM tag. Each of the 12 clones only produced one ERM tag-fused RNA sequence, indicating that each clone contains a single ERM integration. Among these estrogen-independent growth mutants (EIGM), EIGM-1 was a loss-of-function mutation for p27, which was characterized in this study. The other 11 clones included 1 small GTPase (EIGM-2–EIGM-4), 2 components of the Wnt signaling pathway (EIGM-5 and EIGM-6), 1 transcription factor (EIGM-7), 1 Golgi protein (EIGM-8), 1 bone marrow stromal cell antigen (EIGM-9), 1 enolase (EIGM-10), and 2 hypothetical proteins (EIGM-11 and EIGM-12).

Disruption of the single p27kip1 allele in T47D cells results in estrogen-independent growth. The sequence of EIGM-1 matched the second exon of the p27 gene; thus, it was designated as T47DtTA/p27m. Bromodeoxyuridine (BrdUrd) labeling assay confirmed the estrogen-independent growth feature of T47DtTA/p27m cells. About 93% T47DtTA parent cells cultured in the estrogen-free medium were arrested at G1 and <0.3% cells were labeled with BrdUrd. In contrast, only 66% T47DtTA/p27m cells cultured in the estrogen-free medium were in G1 and as many as 8.6% T47DtTA/p27m cells were labeled with BrdUrd, which were similar to T47DtTA and T47DtTA/p27m cells cultured in the growth medium with complete serum (Fig. 1A).

The p27 gene contains two exons separated by a 511-bp intron. Exons 1 and 2 encode for the NH2-terminal 158 and the COOH-terminal 40 (159–198) amino acid residues, respectively (Fig. 1B). PCR and sequence analyses revealed that the ERM integration site was between bp 174 and 175 of intron 1 (Fig. 1B and C). In the RT-PCR product from T47DtTA/p27m cells, the ERM tag was fused in-frame with the second exon of p27, indicating that the 337-bp intron sequence between the splice donor site of the ERM tag and the second exon of p27 was excised during RNA processing (Fig. 1B). Accordingly, the inserted ERM promoter generated a 10-kDa protein that was recognized by antibodies against p27 COOH terminus and ERM tag, indicating that this fusion protein contains ERM tag (62 amino acids) and the COOH-terminal 40 amino acids of p27 (p27-C40; Fig. 1D and data not shown). The p27 NH2-terminal antibody recognized a smaller and less abundant protein in T47DtTA/p27m cells than the full-length p27 in T47DtTA cells (Fig. 1D). Because this smaller p27 protein was not detectable by the p27 COOH-terminal antibody and its apparent molecular mass (~10 kDa) matched the predicted size translated from exon 1 and its extending intron sequence, we concluded that ERM insertion in the p27 intron interfered the splicing between exons 1 and 2. This resulted in a truncated p27 protein with the NH2-terminal 158 amino acids and the COOH-terminal 21 amino acids encoded by the extended intron sequence before meeting the TAA stop codon at 63 to 65 bp of intron 1 (Fig. 1B and D). This NH2-terminal protein was designated as p27-N158. Interestingly, no wild-type p27 allele and full-length p27 protein were detected in T47DtTA/p27m cells by PCR and immunoblotting (Fig. 1C and D), indicating that T47D cells only contain a single p27 wild-type allele and T47DtTA/p27m cells only contain a disrupted p27 allele.

T47DtTA/p27m cells are resistant to antiestrogens although they still express functional ER{alpha}. To test whether disruption of p27 reduces the antiproliferative effects of antiestrogens, we compared cell proliferation rates of T47DtTA/p27m cells and T47DtTA cells treated with 4-OH-tamoxifen and ICI 182780 for 4 days. The vehicle-treated T47DtTA and T47DtTA/p27m cells exhibited similar S-phase fractions (~10%), whereas 4-OH-tamoxifen treatment reduced the S-phase fraction of T47DtTA cells to ~3%. However, 4-OH-tamoxifen treatment only slightly decreased the S-phase fraction of T47DtTA/p27m cells to 7%. ICI 182780 treatment diminished the S-phase fraction of T47DtTA cells to ~3%, but only partially reduced the S-phase fraction of T47DtTA/p27m cells to ~5.5%, indicating that T47DtTA/p27m cells were also less sensitive than T47DtTA cells to ICI 182780 (Fig. 2A ). Consistent results were also obtained from cell growth of T47DtTA and T47DtTA/p27m cells cultured in growth medium containing vehicle, 4-OH-tamoxifen, or ICI 182780 and in estrogen-free medium with CSFCS (Fig. 2B). To validate the specific role of p27 in these experiments, we restored stable p27 expression in T47DtTA/p27m cells by retroviral infection. The restored p27 was a poly-histidine–tagged bigger protein compared with the endogenous p27 in T47DtTA cells (Fig. 2B). Restoration of p27 in T47DtTA/p27m/+p27 cells rescued the growth-inhibitory sensitivities of these cells to 4-OH-tamoxifen, ICI 182780, and CSFCS (Fig. 2B). These results indicate that p27 function is required for T47D cells to respond to the antiproliferative effects of antiestrogens.


Figure 2
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  		Figure 2. T47DtTA/p27m cells are resistant to antiestrogens although they express functional ER{alpha}. A, T47DtTA and T47DtTA/p27m cells in growth medium were treated with vehicle (Veh), 4-OH-tamoxifen (Tam), or ICI 182780 (ICI) for 4 d. The mean of the S-phase fraction was calculated from four assays of flow cytometry. ***, P < 0.001, unpaired t test. B, p27 restoration in T47DtTA/p27m cells rescues their sensitivity to antiestrogens. Immunoblotting assay with the p27 COOH-terminal antibody detected p27 in T47DtTA cells, the truncated p27-C40 in T47DtTA/p27m, and the His-tagged p27 (p27-H) and p27-C40 in T47DtTA/p27m/+p27 (+p27) cells (top). For growth assay (bottom), T47DtTA, T47DtTA/p27m, and T47DtTA/p27m/+p27 cells (105 per well; n = 4) were cultured in medium containing 10% CSFCS overnight and then changed to either medium containing 10% FCS and vehicle, 4-OH-tamoxifen, or ICI 182780 or medium with 10% CSFCS. Cells were cultured for 4 d before harvesting and counted. Columns, mean fold increase in cell number; bars, SD. *, P < 0.05; **, P < 0.01, one-way ANOVA. C, photographs of crystal violet–stained colonies formed from T47DtTA and T47DtTA/p27m cells treated with vehicle, 4-OH-tamoxifen, and ICI 182780 (top). Note the significantly larger and more numerous colonies formed from T47DtTA/p27m cells than T47DtTA cells treated with 4-OH-tamoxifen and ICI 182780. Bottom, quantitative data of colony formation assays in the top. For vehicle- and 4-OH-tamoxifen–treated cells, colonies >1 mm in diameter were counted. For ICI 182780–treated cells, colonies >0.5 mm were counted. Columns, mean colony numbers in eight 1-cm2 areas of culture wells from two repeat experiments; bars, SD. **, P < 0.0; ***, P < 0.001, unpaired t tests. D, immunoblotting analysis of ER{alpha} in T47DtTA and T47DtTA/p27m cells cultured in growth medium and PRB and PRA in T47DtTA and T47DtTA/p27m cells treated with vehicle, 17ß-estradiol (E2), and ICI 182780 in estrogen-free medium. Note that the PRs were increased by estradiol treatment and reduced by ICI 182780 treatment in both types of cells. Protein loading amounts were reflected by immunoblotting analysis of ß-actin.

 
To examine the long-term effects of antiestrogens on T47DtTA/p27m cell survival and growth, we compared the colony formation capability of T47DtTA/p27m cells with that of T47DtTA cells over a 3-week culture period. In growth medium, T47DtTA/p27m and T47DtTA cells formed colonies equally well, giving ~45 colonies/cm2. 4-OH-Tamoxifen treatment significantly reduced the density of colonies formed from T47DtTA cells to 15 colonies/cm2. In contrast, 4-OH-tamoxifen treatment did not significantly change the colony formation capability of T47DtTA/p27m cells (Fig. 2C). ICI 182780 treatment profoundly reduced both the number and size of the colonies formed from T47DtTA cells. However, ICI 182780 reduced only the colony size of T47DtTA/p27m cells but not their total number of colonies (Fig. 2C). These results show that T47DtTA/p27m cells are fully resistant to 4-OH-tamoxifen and partially resistant to ICI 182780.

It is a possibility that loss of ER{alpha} causes breast cancer cells to become insensitive to antiestrogens. However, immunoblotting analysis revealed that ER{alpha} levels in T47DtTA and T47DtTA/p27m cells were comparable. The estradiol-induced or ICI 182780–inhibited transcriptions of PRA and PRB from the estrogen-responsive PR gene also were comparable in these cells (Fig. 2D). Thus, the antiestrogen resistance observed in T47DtTA/p27m cells is not due to a loss of ER{alpha} expression or function.

The p27-N158 and p27-C40 proteins are loss-of-function mutants. Most p27 immunoreactivity was detected in the nucleus of T47DtTA cells by p27 NH2-terminal antibody, whereas the p27-N158 immunoreactivity was detected in both cytoplasm and nucleus of T47DtTA/p27m cells with the same antibody (Fig. 3A ). Immunoblotting analyses further identified p27 in both cytoplasmic and nuclear fractions of T47DtTA cells; on the contrary, the mutant p27-N158 was mainly found in the cytoplasmic fraction of T47DtTA/p27m cells (Fig. 3B, top). The latter observation is consistent with the absence of p27 nuclear localization sequence (amino acids 159–169) in p27-N158 (Fig. 1B). These results indicate that although p27-N158 contains the NH2-terminal domains for binding cyclins and CDKs, it mainly stays in the cytoplasm and does not function as a nuclear CDK inhibitor.


Figure 3
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  		Figure 3. Subcellular localization and function of p27, p27-N158, and p27-C40 proteins and insensitive inhibition of the CDK2 kinase activity in T47DtTA/p27m cells by 4-OH-tamoxifen, ICI 182780, and CSFCS. A, immunocytofluorescence labeling of T47DtTA and T47DtTA/p27m cells with antibodies against p27 NH2 terminus ({alpha}-P27-N) and p27 COOH terminus ({alpha}-P27-C). Note the cytoplasmic localization of immunoactivity in T47DtTA/p27m cells detected by both antibodies. 4',6-Diamidino-2-phenylindole (DAPI) staining was used to show cell nuclei. B, immunoblotting analysis of cytosol and nuclear fractions of T47DtTA and T47DtTA/p27m cells using p27 NH2-terminal (top) and p27 COOH-terminal (bottom) antibodies. The subcellular fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Chemical). C, immunoblotting analysis of T47DtTA and T47DtTA/p27m cells using mixed p27 NH2-terminal and p27 COOH-terminal antibodies after cells were treated with or without tetracycline (Tet). All samples were assayed in the same blot; the image of the left two lanes and the image of the right two lanes were aligned together after the image of two unused lanes between them was deleted. ß-Actin was assayed as a loading control. D, treatment of T47DtTA and T47DtTA/p27m cells with vehicle, 4-OH-tamoxifen, ICI 182780, or CSFCS (estrogen-free medium) and immunoblotting assays of p27, p27-N158, cyclin E, CDK2, 32P-histone H1 (32P-H1), hyperphosphorylated Rb (ppRb), hypophosphorylated Rb (ppRb), hyperphosphorylated p130 (pp130), hypophosphorylated p130 (p130), and ß-actin control. For CDK2 kinase activity assay, the cyclin E-CDK2 complexes were immunoprecipitated with a cyclin E antibody from equal amounts of cell lysates. CDK2 kinase activity was assayed by using [{gamma}-32P]ATP and histone H1 as substrates. Relative band intensity (Rel. Int.) of the phosphorylated histone H1 in each lane is indicated by setting the first lane as 1. For immunoblotting analyses of the phosphorylated Rb and p130, the ratios of hyperphosphorylated Rb and pp130 to hypophosphorylated Rb and p130 are indicated for each lane, respectively. Note that the increases in hypophosphorylated Rb and p130 were observed only in T47DtTA cells but not in T47DtTA/p27m cells after these cells were treated with 4-OH-tamoxifen, ICI 182780, and CSFCS.

 
The nuclear p27 in T47DtTA cells was also detected by the p27 COOH-terminal antibody. Interestingly, the p27-C40, detected by the same antibody, was mainly found in the cytoplasm of T47DtTA/p27m cells, although weaker immunoreactivity was also detected in their nuclei (Fig. 3A). Immunoblotting analyses confirmed that p27-C40 was distributed in both cytoplasmic and nuclear fractions of T47DtTA/p27m cells (Fig. 3B, bottom). Because p27-C40 expression is controlled by the tetracycline-off ERM promoter, we examined the effect of p27-C40 on cell proliferation by treating cells with tetracycline. The p27-C40 level was significantly reduced in T47DtTA/p27m cells on tetracycline treatment (Fig. 3C). However, the distribution of cell fractions in all proliferation phases, determined by flow cytometry, was identical between vehicle- and tetracycline-treated T47DtTA/p27m cells (data not shown). These results indicate that change of p27-C40 concentration does not affect cell proliferation.

p27 deficiency reduces the inhibitory effects of antiestrogens on CDK2 activity. In estrogen-dependent breast cancer cells, antiestrogens induce p27, inhibit cyclin E-CDK2 activity, reduce Rb and Rb-related p130 hyperphosphorylation, suppress E2F, and arrest cell cycle at G1 (16). In T47DtTA cells, 4-OH-tamoxifen induced a slight, and ICI 182780 and estrogen-free condition induced a significant, p27 increase. In T47DtTA/p27m cells, these treatments failed to induce p27-N158 levels, suggesting that antiestrogens or estrogen-free conditions do not induce p27 promoter activity in the estrogen-independent T47DtTA/p27m cells. The level of p27-C40 was decreased when T47DtTA/p27m cells were cultured in estrogen-free medium, due presumably to lower ERM promoter activity under this culture condition (Fig. 3D and data now shown). Cyclin E and CDK2 levels were identical between T47DtTA and T47DtTA/p27m cells under all examined conditions (Fig. 3D).

To examine the effect of p27 mutation on the kinase activity of cyclin E-CDK2 complexes, cyclin E was immunoprecipitated from cell extracts with equal protein amount and the kinase activity of precipitates was assayed using histone H1 as substrate. The cyclin E–associated CDK2 kinase activity was reduced by 33% and 66%, respectively, when T47DtTA cells were treated with 4-OH-tamoxifen and ICI 182780. When T47DtTA cells were cultured in estrogen-free medium, the cyclin E–associated CDK2 kinase activity was reduced by 87%. In contrast, the cyclin E-CDK2 kinase activity in T47DtTA/p27m cells was not significantly reduced with 4-OH-tamoxifen and only reduced by 37% and 57% with ICI 182780 and estrogen-free medium (Fig. 3D). In agreement with the increase of CDK2 activity, the ratios of hyperphosphorylated Rb to hypophosphorylated Rb were increased in T47DtTA/p27m cells treated with 4-OH-tamoxifen, ICI 182780, and estrogen-free medium compared with the ratios of hyperphosphorylated Rb to hypophosphorylated Rb in T47DtTA cells receiving the same treatments (Fig. 3D). Similarly, the ratios of hyperphosphorylated p130 to hypophosphorylated p130 in T47DtTA/p27m cells treated with 4-OH-tamoxifen, ICI 182780, and estrogen-free medium were higher than the ratios of hyperphosphorylated p130 to hypophosphorylated p130 in T47DtTA cells (Fig. 3D). The increase in these ratios was mainly due to the decrease in the hypophosphorylated inhibitory forms of Rb and p130. These results indicate that p27 deficiency desensitizes the inhibition of CDK2 activity by antiestrogens and estrogen depletion, causing sustained hyperphosphorylation and inactivation of tumor suppressors including hypophosphorylated Rb and p130, even in the presence of antiestrogens or in the absence of estrogen.

Loss of p27 stimulates AIB1 phosphorylation and E2F1 transcriptional activity. The elevated CDK2 activity is known to activate E2F transcriptional activity, which plays an essential role in G1-S transition of the cell cycle. To measure E2F1 activity, we transfected T47DtTA and T47DtTA/p27m cells with an E2F1-responsive luciferase reporter and measured the luciferase activity. The reporter activity was ~2-fold higher in T47DtTA/p27m cells compared with T47DtTA cells when these cells were treated with vehicle, 4-OH-tamoxifen, ICI 182780, or estrogen-free medium (Fig. 4A ). These results indicate that p27 deficiency significantly stimulates E2F1 activity in T47DtTA/p27m cells.


Figure 4
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  		Figure 4. p27 deficiency enhances AIB1 phosphorylation and increases the transcriptional activity of E2F1 and AIB1. A, T47DtTA/p27m cells have higher E2F1 activity compared with T47DtTA cells. E2F1 activity was measured by transfecting cells with an E2F1 luciferase reporter. Transfected cells were cultured in growth medium containing vehicle, tamoxifen, and ICI 182780 or in medium with 10% CSFCS for 2 d. Transfection efficiency was normalized to ß-galactosidase activity, which was expressed by cotransfecting cells with the pRSV-ß-gal vector. Data are presented by setting relative average luciferase activity in CSFCS-treated T47DtTA cells as 1 unit. B, immunoblotting analysis showing comparable AIB1 levels in T47DtTA and T47DtTA/p27m cells. Immunoblotting analysis of ß-actin serves as a loading control. C, p27 deficiency and 17ß-estradiol (E2) enhance AIB1 phosphorylation. T47DtTA and T47DtTA/p27m cells were cultured in the presence or absence of 17ß-estradiol for 1 h in medium with 10% CSFCS. Total AIB1 was immunoprecipitated with AIB1 antibody and then subjected to immunoblotting analysis with antibodies specific to the phosphorylation sites as indicated. These antibodies were developed and affinity purified as described (29). Note that the total amount of precipitated AIB1 is similar among groups but the different sites are differentially phosphorylated. D, mutation of specific phosphorylation sites impairs AIB1 coactivator activity for E2F1-mediated target gene transcription. Cells were transfected with E2F1, E2F1-responsive luciferase reporter, and AIB1 or its mutants as indicated. Luciferase activity was assayed as described in Materials and Methods. Columns, mean (n = 4; with the first column set as 1 unit); bars, SD. *, P < 0.05; **, P < 0.01, one-way ANOVA.

 
AIB1 is a strong coactivator for E2F1 and plays an important role in breast cancer proliferation and antiestrogen resistance (22, 23, 36, 37). Because AIB1 cellular concentration and phosphorylation levels determine its coactivator activity, we assessed if p27 deficiency would alter AIB1 protein level and/or phosphorylation status using specific antibodies detecting total AIB1 and its individual phosphorylated sites known to be important for its coactivator activity (29). Whereas total AIB1 remained unchanged in T47DtTA/p27m cells compared with T47DtTA cells (Fig. 4B and C), its phosphorylation levels on five of the six known sites, with the exception of Ser543, were significantly enhanced in T47DtTA/p27m cells, including phosphorylated Thr24 (pT24) and phosphorylated Ser505 (pS505), pS857, pS860, and pS867 in the absence of estrogen treatment (Fig. 4C). Interestingly, 17ß-estradiol treatment stimulated the phosphorylation of these sites in T47DtTA cells but not in T47DtTA/p27m cells (Fig. 4C), suggesting that the phosphorylation levels of these sites induced by p27 deficiency are similar to the levels induced by estrogen in T47DtTA cells.

Next, we analyzed the effect of each AIB1 phosphorylation site on E2F1 reporter by comparing the coactivator activity of AIB1 with its mutants containing a threonine (T) or serine (S) to alanine (A) mutation. These mutant and wild-type AIB1 vectors showed similar expression levels in MCF-7 breast cancer cells. MCF-7 cells were used because they gave a better efficiency than T47D cells when multiple plasmids were cotransfected. Our experiments showed that expression of E2F1 or AIB1 alone had limited stimulation on E2F1 reporter activity, but coexpression of E2F1 and AIB1 robustly potentiated E2F reporter activity (Fig. 4D). Expression of the T24A, S505A, S543A, and S860A AIB1 mutants also coactivated E2F1 activity as wild-type AIB1 did. Nevertheless, coactivator activities were partially impaired for the S867A mutant and completely diminished for the S857A mutant (Fig. 4D). These results suggest that phosphorylation on two of the six sites of AIB1 has a positive effect on E2F1-mediated transcription. Because disruption of p27 increased the phosphorylation on five of the six known sites including pS857 and pS867, the hyperphosphorylated AIB1 in T47DtTA/p27m cells should serve as a stronger coactivator for E2F1 compared with the hypophosphorylated AIB1 in T47DtTA cells.

Disruption of p27 increases Akt activity. The adaptor protein Gab2 is a direct E2F target (30). Indeed, Gab2 was significantly increased in T47DtTA/p27m cells compared with T47DtTA cells; restoration of p27 caused a reversible decrease in Gab2 in T47DtTA/p27m/+27 cells (Fig. 5A ). The E2F-induced Gab2 plays an essential role in mediating E2F-dependent activation of the PI3K/Akt signaling pathway in U-2OS osteosarcoma cells (30). To address whether p27 deficiency–induced hyperactive E2F and overexpressed Gab2 would also potentiate Akt activation in breast cancer cells, we assayed total Akt and active Akt, the phospho-Ser473-Akt (pS473-Akt; ref. 38), by immunoblotting. The total Akt was comparable in T47DtTA/p27m and T47DtTA cells (Fig. 5A). However, the pS473-Akt was ~3-fold higher in T47DtTA/p27m cells than in T47DtTA cells. Restoration of p27 in T47DtTA/p27/+27 cells did not change total Akt but decreased active Akt (Fig. 5A). Accordingly, Akt protein immunopurified from T47DtTA/p27m cells showed higher kinase activity than Akt from T47DtTA cells as determined by in vitro kinase assays using glycogen synthase kinase (GSK)-3ß as substrate (ref. 39; Fig. 5A). These results show that inactivation of p27 in T47D cells causes superactivation of Akt, probably by enhancing the transcriptional capability of E2F.


Figure 5
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  		Figure 5. Inactivation of p27 increases Gab2 protein and Akt activity, cell motility, and cell-invasive ability. A, p27 deficiency increases Gab2 protein and Akt activity. Gab2 levels in T47DtTA, T47DtTA/p27m, and T47DtTA/p27m/+27 (+27) cells were assayed by immunoblotting. Total Akt and phosphorylated active Akt in these cells were detected by immunoblotting with respective Akt antibody and phosphorylation site–specific antibody. Akt activity in T47DtTA and T47DtTA/p27m cells was assayed using GSK-3ß as substrate and the phosphorylated GSK-3ß (p-GSK) was detected by immunoblotting with a phospho-GSK-3ß–specific antibody. The ß-actin assay served as a loading control. B, measurement of cell migration. T47DtTA and T47DtTA/p27m cells were cultured on plates coated with fibronectin (FN), laminin (LN), collagen I (Col I), or collagen IV (Col IV), and cell migration was traced by using blue fluorescence beads. Individual cells (arrows) were stained with rhodamine-phalloidin. Dark areas indicate cell migration tracks. Cells migrated faster on fibronectin and collagen IV. C, measurement of migration areas. Cell migration tracks of 25 to 30 cells were analyzed by using NIH image software. Columns, average relative migration area; bars, SD. The migration areas of T47DtTA/p27m cells on all coated matrices examined are significantly bigger compared with T47DtTA cells (P < 0.01, unpaired t test). D, cell invasion assay. The invasive capability of T47DtTA, T47DtTA/p27m, and T47DtTA/p27m/+27 in the Matrigel was measured as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD.

 
Inactivation of p27 promotes cell mobility and invasion. The PI3K/Akt signaling pathway plays a central role in cancer cell motility, invasion, and metastasis (31, 32). To assess the effects of p27 deficiency–triggered Akt activation on cell motility, we measured the extent of individual cell migration on extracellular matrix proteins by using blue fluorescent beads to determine the track area cells had moved through in 18 h. On culture plates coated with each of the four extracellular matrix proteins, the track area of T47DtTA/p27m cells was significantly larger than the track area of T47DtTA cells (Fig. 5B). Quantitative analysis of the track areas revealed that T47DtTA/p27m cells migrated 2.1-, 4.6-, 4.5-, and 3-fold faster than T47DtTA cells on fibronectin-, laminin-, collagen I–, and collagen IV–coated culture plates, respectively (Fig. 5C). These results indicate that inactivation of p27 in T47D cells remarkably promotes cell motility.

We also evaluated the capability of T47DtTA, T47DtTA/p27m, and T47DtTA/p27m/+p27 cells to invade through the extracellular matrix by using Matrigel Invasion Chambers. The average number of T47DtTA/p27m cells that invaded through the Matrigel layer increased >3-fold compared with T47DtTA and T47DtTA/p27m/+p27 cells (Fig. 5D). These results indicate that p27 deficiency significantly enhances the invasive capability of breast cancer cells.

T47DtTA/p27m and T47DtTA cells showed similar cell adhesion capability on fibronectin-, laminin-, collagen I–, or collagen IV–coated culture plates (data not shown). Immunoblotting and immunocytochemistry revealed that the levels and distribution patterns of E-cadherin, an epithelial marker for mammary epithelial association (40), were similar between T47DtTA and T47DtTA/p27m cells (data not shown). These results indicate that p27 deficiency does not alter E-cadherin expression and location as well as mammary epithelial cell-cell interaction.

I.v. injection of p27-deficient T47DtTA/p27m cells induces lung metastasis in ovariectomized nude mice. T47D cells are estrogen-dependent human breast cancer cells, which can only form nonmetastatic local tumors in ovariectomized nude mice treated with estrogen (35). To test whether p27 deficiency in estrogen-dependent breast cancer cells could promote tumor formation in an ovarian hormone–independent manner, we compared tumor development and progression of T47DtTA/p27m cells with T47DtTA cells in ovariectomized nude mice. Both T47DtTA and T47DtTA/p27m cells developed local tumors in the mammary fat pats of all nude mice (n = 6) treated with estradiol pellets, but failed to develop tumors in the mammary fat pads of nude mice treated with placebo (Fig. 6A and data not shown). Histologic examination revealed that T47DtTA tumors had a smooth surface and a relatively tight cell-cell association (Fig. 6A). In contrast, the edge of T47DtTA/p27m tumors frequently protruded into the surrounding stromal tissues (Fig. 6A). These results suggest that loss of p27 function may make T47DtTA/p27m tumors more invasive.


Figure 6
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  		Figure 6. Primary and metastatic tumor formation in ovariectomized nude mice. A, top, primary tumors (arrows) formed from T47DtTA and T47DtTA/p27m cells in nude mice with 17ß-estradiol pellets. Bottom, images of H&E-stained tumor sections prepared from T47DtTA and T47DtTA/p27m tumors. Note the different morphologies of the tumor edges (arrows) in the two types of tumors. Bar, 50 µm. B, photographs of lungs from different ovariectomized nude mice receiving T47DtTA/p27m cells from their tail veins. Arrows, visible metastatic lung tumors. C, H&E-stained lung sections prepared from ovariectomized nude mice receiving i.v. injection of T47DtTA and T47DtTA/p27m cells. Right, top, an invasive big lung tumor is outlined; bottom, larger images of the boxed areas in the top. Bar, 500 µm (top) and 50 µm (bottom). D, immunohistochemical staining of K8 (brown color) on lung sections prepared from ovariectomized nude mice injected with T47DtTA or T47DtTA/p27m cells as indicated. Bar, 50 µm.

 
To address if the local tumors formed from T47DtTA/p27m cells might develop systemic metastasis, we examined the lung and other internal organs of mice bearing T47DtTA/p27m tumors in their mammary fat pats. However, we did not find any metastasis that developed in any secondary organs, suggesting that T47DtTA/p27m tumors formed in the mammary gland are unable to accomplish the entire process of distant metastasis.

To determine whether p27 deficiency–induced cellular alterations could potentiate metastasis of breast cancer cells after intravasation, we compared the capability of T47DtTA/p27m cells with that of T47DtTA cells to form metastatic tumors in the lung using a well-established experimental metastasis method (41). T47DtTA cells did not develop any tumors in the lung of ovariectomized nude mice (n = 5) 3 weeks after injection i.v. (Fig. 6C). In contrast, all of the seven ovariectomized nude mice developed lung tumors 3 weeks after receiving T47DtTA/p27m cells i.v. In these mice lacking ovarian steroids, many tumors were visible on the lung surfaces, and big invasive tumors were observed on lung sections (Fig. 6B and C). In these tumor cells, strong immunoreactivity of p27-C40 was detected in the cytoplasm, which was similar to that seen in T47DtTA/p27m cells in Fig. 3A. In addition, the mammary epithelial marker K8 was also detected in the lung tumor cells of nude mice injected with T47DtTA/p27m cells, but not in the lung of nude mice injected with T47DtTA cells (Fig. 6D), indicating that these metastatic tumors originated from T47DtTA/p27m cells. These results show that loss of p27 in T47D estrogen-dependent breast cancer cells is sufficient to permit metastatic tumor formation in the lung in an ovarian hormone–independent manner, once these tumor cells enter the circulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Development of estrogen independence and tamoxifen resistance is a major problem in breast cancer endocrine therapy. Characterization of the genetic changes responsible for the transition from estrogen dependence to estrogen independence and from tamoxifen responsiveness to tamoxifen resistance is essential for understanding the mechanisms for this key step in progression. In this study, we found that the estrogen-dependent T47D cells contain only one p27 allele, suggesting that p27 is already decreased in the original T47D tumor. Our genome-wide screening using ERM identified the p27-deficient T47DtTA/p27m cells that still express functional ER{alpha} but grow in an estrogen-independent manner and are fully resistant to tamoxifen. These observations recapitulate the similar features of ER{alpha}-positive breast cancers that progress from tamoxifen sensitive to tamoxifen resistant and are consistent with an important role of p27 reduction and cytoplasmic retention in breast cancer initiation and progression (13–15, 17–19). Because our results show that disruption of p27 is sufficient to permit estrogen-dependent and tamoxifen-sensitive breast cancer cells to grow in the absence of estrogen and in the presence of tamoxifen and that these growth phenotypes can be rescued by p27 restoration, we conclude that functional p27 is required for tamoxifen-mediated inhibition of ER{alpha}-positive breast cancer growth.

T47DtTA/p27m cells also exhibited a partial resistance to ICI 182780 compared with T47DtTA cells. ICI 182780 only reduced the size of T47DtTA/p27m cell colonies and inhibited 37% of CDK2 activity. Previous studies have shown that both p27 and p21cip1 can contribute to antiestrogen-mediated cell cycle arrest in breast cancer cells (18). Our analyses also reveal that p21 is more obviously induced by ICI 182780 in T47DtTA/p27m cells than in T47DtTA cells and more cyclin D1-CDK4 and cyclin E-CDK2 were associated with p21cip1 in T47DtTA/p27m cells (data not shown). Therefore, the partial ICI 182780 resistance of T47DtTA/p27m cells may be accredited to the compensatory role of p21cip1 when p27 function is lost.

E2F1 transcriptional activity is significantly elevated in T47DtTA/p27m cells because p27 deficiency activates CDK2, leading to Rb phosphorylation and E2F1 activation. Recently, AIB1 has been identified as a major coactivator for E2F1; E2F1 interacts with AIB1 NH2 terminus and recruits AIB1 to the promoter of E2F1 target gene (22). Other studies also have shown that growth factors, cytokines, and estrogen can stimulate AIB1 phosphorylation at specific sites and AIB1 phosphorylation usually alters AIB1 coactivator activity for steroid receptors (29, 42). Nevertheless, the mechanisms leading to AIB1 phosphorylation are complex and many kinases including p38, I{kappa}B kinases, extracellular signal–regulated kinase, c-jun NH2-terminal kinase, and GSKs have been shown to phosphorylate AIB1 (29). Because CDK2 and Akt are stimulated in p27-deficient T47DtTA/p27 cells, these two kinases might also play a role in AIB1 phosphorylation. Our data show that loss of p27 in T47DtTA/p27m cells enhances AIB1 phosphorylation on five of the six mapped sites, and the phosphorylation on four of these five sites was also enhanced by estrogen in T47DtTA cells (Fig. 5C), suggesting that p27 deficiency causes an AIB1 phosphorylation pattern similar to that caused by estrogen. Importantly, mutation of two of these sites (S857 and S867) either attenuated or diminished AIB1 coactivator activity for E2F1. These results indicate that the phosphorylated AIB1 is a better coactivator for E2F1. Thus, p27 deficiency–induced AIB1 phosphorylation further potentiates E2F1 target gene transcription following CDK2 activation and Rb phosphorylation. This double enhancement of E2F1 activity may play a crucial role in breast cancer progression into estrogen independence and tamoxifen resistance.

Intriguingly, T47DtTA/p27m cells can grow in estrogen-free medium but are unable to develop tumors in the mammary fat pads of ovariectomized nude mice with or without tamoxifen treatment, suggesting that tumor formation in the mammary fat pads in the absence of ovarian hormones is more difficult than cell growth in estrogen-free medium for T47DtTA/p27m cells. With estrogen replacement, both T47DtTA and T47DtTA/p27m cells developed solid tumors in the mammary fat pads. Although T47DtTA/p27m tumors are morphologically more invasive, both T47DtTA and T47DtTA/p27m tumors formed in the mammary fat pads are not metastatic, suggesting that p27 deficiency alone is not sufficient for these tumor cells to progress into fully metastatic cancer cells.

The next question is why T47DtTA/p27m cells lacking p27, but not T47DtTA cells with p27, develop lung metastasis when injected into the circulation of ovariectomized mice. To develop lung metastasis, tumor cells, after intravasation or injection into the circulation, must attach to the endothelium of small lung vessels, migrate or invade through the vessel wall, and form tumors in the lung (41). Our analyses show that T47DtTA and T47DtTA/p27m cells have similar adhesion capability to extracellular matrix proteins, suggesting that p27 deficiency does not change cell adhesion properties and the increase in T47DtTA/p27m cell metastatic potential is unlikely due to the change of endothelial attachment. On the other hand, cell migration and invasion abilities are correlated with metastasis. Our data show that p27 deficiency in T47DtTA/p27m cells dramatically promotes their motility and invasiveness compared with T47DtTA cells. Thus, p27 deficiency–induced increase in cell motility and invasion may be responsible for the ability of T47DtTA/p27m cells to develop lung metastasis.

The mechanisms for p27 deficiency–induced cell motility, invasiveness, and lung metastasis may be very complex. We showed that T47DtTA/p27m cells have an enhanced AIB1 phosphorylation, which makes AIB1 a stronger coactivator for E2F1. Previous studies have established that E2F1 strongly activates Akt through a direct up-regulation of Gab2 expression (30). In this study, p27 deficiency in T47DtTA/p27m cells indeed increases E2F1 transcriptional activity, Gab2 protein, and Akt activation following AIB1 phosphorylation. Akt plays a central role in cancer cell motility, invasion, and metastasis (31, 32). Therefore, p27 deficiency–enhanced AIB1 phosphorylation may significantly contribute to cell motility, invasion, and metastasis by activating Akt. This interpretation is consistent with the lower Akt activity and less lung metastasis of the mammary gland tumors induced by oncogenes and carcinogens in AIB1-null mice compared with wild-type mice (27, 28).

Furthermore, it has been shown that overexpression of a p27 cytosolic mutant increases total Akt by inhibiting its turnover (43). Nevertheless, it is unlikely that the disrupted p27 fragments in T47DtTA/p27m cells can play the same role that the full-length cytosolic p27 mutant does because there is no change in total Akt in T47DtTA/p27m cells. In addition, p27 deficiency may release its direct inhibition on cell motility. Although p27 has been reported as both an inhibitor and a stimulator of cell migration (44–47), a recent article has suggested that the controversial observations about the role of p27 in cell motility may be due to the type of cell motility assayed (48). This study showed that p27 binds and impairs the function of the microtubule-destabilizing protein stathmin and thereby plays an inhibitory role in amoeboid cell migration, a type of cell movement used by tumor cells in tissues (48). Disruption of p27 in T47DtTA/p27m cells may impair the direct inhibitory role of p27 in cell motility and tumor cell metastasis. Finally, down-regulation of p27 causes an up-regulation of G protein–coupled receptor 48 (GPR48), and GPR48 can enhance cancer cell invasiveness and metastasis (49). Therefore, multiple pathways may be involved in the p27 deficiency–induced increase in breast cancer cell motility, invasiveness, and metastasis.

Although p27 null mutations are rare, ~50% human tumors exhibit decreased p27. The p27 reduction and cytoplasmic mislocalization correlate with more aggressive phenotypes, including HER2/neu overexpression, high proliferating indices, active invasive behavior, and high mortality (13–15, 50). These observations suggest that p27 abundance inversely correlates with breast cancer progression. However, studies using transgenic mice suggest that a threshold of p27 seems to be required for normal mammary epithelial proliferation and mammary tumorigenesis induced by oncoproteins. For example, p27+/– mice exhibit a faster mammary epithelial growth and mouse mammary tumor virus (MMTV)-neu–induced tumorigenesis, but p27–/– mice exhibit a delayed mammary gland morphogenesis and MMTV-neu–induced tumorigenesis (21). Molecular analysis suggests that certain p27 is required for stabilization of cyclin D1 and assembly and nuclear translocation of cyclin D1-CDK4 complexes. Thus, CDK4 in p27–/– mammary epithelial cells cannot be activated, which in turn suppresses the neu-induced, cyclin D1-dependent mammary epithelial transformation (21). Similarly, cyclin D1 is decreased and CDK4 activity is reduced manyfold in our T47DtTA/p27m cells (data not shown). However, T47DtTA/p27m cells exhibit comparable proliferation as T47DtTA cells when cultured in growth medium. These results indicate that T47DtTA/p27m cells are no longer dependent on cyclin D1-CDK4 for survival and growth. Although it is unknown whether the T47D parent tumor has acquired cyclin D1-CDK4–independent growth, the increase in cyclin E-CDK2 activity after loss of p27 in T47DtTA/p27m cells is cyclin D1-CDK4 independent and should be responsible for the antiestrogen insensitivity of these cells.

Based on this and previous studies discussed above, the effect of p27 gene dosage on breast cancer is likely dependent on the specific stages of tumor initiation and progression. It seems reasonable to propose that p27 reduction in the mammary epithelium and early stages of tumorigenesis may promote cell proliferation and accelerate hormone-, oncoprotein-, and carcinogen-induced transformation while still maintaining cyclin D1 and CDK4 activity. Once these transformed cells acquire a cyclin D1-CDK4–independent growth feature, a further reduction or loss or mislocalization of p27 may facilitate breast tumor cells to develop more aggressive cancer phenotypes including estrogen-independent growth, antiestrogen insensitivity, and metastasis. During this process, p27 insufficiency– or p27 deficiency–induced increase in AIB1, E2F1, Gab2, and Akt activities and decrease in p27 inhibitory role in cell motility may have a major contribution to breast cancer progression.


   Acknowledgments
 
Grant support: NIH grants DK58242, CA119689, CA112403 (J. Xu), and CA84208 (Z. Songyang) and American Cancer Society Research Scholar Award RSG-05-082-01 (J. Xu).

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

We thank Lan Liao for experimental assistance.

Received 1/ 8/07. Revised 5/25/07. Accepted 6/26/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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