Elevated expression of Akt-1 (PKBα) has been noted in a significant percentage of primary human breast cancers. Another frequent event in the genesis of human breast cancers is amplification and overexpression of the ErbB-2 receptor tyrosine kinase, an event which is associated with activation of Akt-1. To directly assess the importance of Akt-1 activation in ErbB-2 mammary tumor progression, we interbred separate strains of transgenic mice carrying mouse mammary tumor virus/activated Akt-1 and mouse mammary tumor virus/activated ErbB-2 to derive progeny that coexpress the transgenes in the mammary epithelium. Female transgenic mice coexpressing activated Akt-1 and ErbB-2 develop multifocal mammary tumors with a significantly shorter latency period than mice expressing activated ErbB-2 alone. This dramatic acceleration of mammary tumor progression correlates with enhanced cellular proliferation, elevated Cyclin D1 protein levels, and phosphorylation of retinoblastoma protein. These bitransgenic mammary tumors also exhibit lower levels of invasion into the surrounding tissue and more differentiated phenotypes. Consistent with these observations, female mice coexpressing activated Akt-1 and ErbB-2 developed significantly fewer metastatic lesions than the activated ErbB-2 strain alone. Taken together, these observations suggest that activation of Akt-1 during ErbB-2-induced mammary tumorigenesis may have opposing effects on tumor growth and metastatic progression.

ErbB-2 is a member of the epidermal growth factor receptor family of receptor tyrosine kinases, which consists of four closely related type-1 receptor tyrosine kinases, including: (a) epidermal growth factor receptor; (b) ErbB-2 (Neu and HER-2); (c) ErbB-3 (HER-3); and (d) ErbB-4 (HER-4; Ref. 1). The importance of ErbB-2 in primary human breast cancer is highlighted by studies demonstrating that 20–30% of human breast cancers express elevated levels of ErbB-2 because of genomic amplification of the ErbB-2 proto-oncogene (2). Significantly, this amplification and subsequent overexpression strongly correlate with a negative clinical prognosis in both lymph node-positive (3) and -negative (4) breast cancer patients. Additional evidence that overexpression of ErbB-2 results in an aggressive tumor type comes from studies showing elevated ErbB-2 expression in many in situ and invasive human ductal carcinomas but not in benign breast disorders, such as hyperplasias and dysplasias (3). Importantly, ErbB-2 overexpression may also be useful as a prognostic treatment marker, because it predicts tamoxifen resistance of the primary tumor (5).

Epidermal growth factor receptor family member signaling entails the formation of homo and hetero-dimers in response to ligand stimulation, resulting in the phosphorylation of specific receptor tyrosine residues (1). These phosphorylated tyrosines then offer docking sites for the SH2 and SH3 (PTB) domains of various signaling molecules, which transduce downstream signals (1). ErbB-2 binds and activates many downstream signaling molecules in this manner, including the phosphatidylinositol-3′-OH kinase (PI-3K; Ref. 6), a lipid kinase that phosphorylates 3′ hydroxyl residues in the inositol rings of certain lipids (7). These phospholipids act as second messengers to recruit and activate downstream targets, including the serine/threonine kinase Akt-1/PKBα. Akt-1, which has two mammalian homologues, Akt-2/PKBβ and Akt-3/PKBγ, possesses a pleckstrin homology domain at its NH2 terminus that specifically binds PI-3K phospholipid products (8). This phospholipid binding targets Akt-1 to the cell membrane, where it is phosphorylated and activated by the PDK1 kinase and a kinase activity termed PDK2 (8). Akt-1 activation has been implicated in a variety of cellular processes, including cell growth and proliferation, protection from apoptosis, and regulation of gene expression at both the transcriptional and translational levels (for a review of Akt-1’s roles, see Ref. 9). Akt-1 is implicated in human cancer progression, because the Akt-1 gene is amplified in human gastric cancers (10), and Akt-1 kinase activity is frequently increased in breast cancers, where it is associated with poor prognosis (11, 12) and resistance to tamoxifen and radiotherapy (13). Furthermore, the tumor suppressor PTEN, a lipid phosphatase that dephosphorylates PI-3K lipid products, negatively regulates PI-3K/Akt-1 signaling (14).

In an effort to directly assess the importance of the PI-3K/Akt-1 signaling pathway in mammary development and tumorigenesis, we generated transgenic mice which express a constitutively activated version of Akt-1 (AktDD) in the mammary epithelium (15). Although this activated Akt-1 did not induce mammary carcinoma, the mouse mammary tumor virus (MMTV)/AktDD animals displayed dramatic defects in mammary gland involution (15). Consistent with our observations, mammary epithelial expression of a membrane-localized version of Akt-1 in transgenic mice results in an identical mammary phenotype (16). However, mammary epithelial coexpression of AktDD with a mutant polyomavirus middle T antigen unable to signal through PI-3K (MTY315/322F) resulted in a dramatic acceleration of mammary tumorigenesis and correlated with reduced apoptotic cell death (15). Furthermore, coexpression of AktDD with MTY315/322F resulted in enhanced phosphorylation of the FKHR forkhead transcription factor and post-transcriptional up-regulation of Cyclin D1 levels (15). Significantly, we did not observe a restoration of metastasis to wild-type levels in this bitransgenic strain (15).

Akt-1 may play important roles in ErbB-2-mediated mammary tumorigenesis, because ErbB-2 can transduce multiple signals through the PI-3K/Akt-1 pathway (17, 18, 19), and Akt-1 kinase activity correlated with ErbB-2/HER-2 overexpression in a study of human breast cancer samples (11). Aside from these studies, initial evidence suggesting a role for Akt-1 activity in ErbB-2-mediated mammary tumorigenesis derives from studies of transgenic mice expressing activated ErbB-2 transgenes in the mammary gland. Transgenic mice carrying altered ErbB-2/Neu or Neu deletion line (NDL) receptors under MMTV control develop multiple mammary tumors which frequently metastasize to the lung (20). Significantly, tumor progression in these strains is specifically associated with elevated levels of tyrosine-phosphorylated ErbB-3, and consistent with these observations, Siegel et al.(20) also noted frequent coexpression of both ErbB-2 and ErbB-3 transcripts in human breast tumors. These results further implicate PI-3K/Akt-1 signaling in ErbB-2-mediated mammary tumorigenesis, because ErbB-3 possesses seven consensus docking sites for PI-3K (21) and has been shown to signal through Akt-1 (22).

If activation of Akt-1 signaling is indeed a critical step in ErbB-2 mammary tumor progression, it is expected that early activation of Akt-1 would accelerate the process of ErbB-2-mediated mammary tumor formation. To test this hypothesis, we have crossed transgenic mice expressing the MMTV/activated Akt-1/AktDD transgene with an MMTV/activated ErbB-2/NDL strain (NDL2–5; Ref. 20) to generate bitransgenic animals. Our results reveal that activation of Akt-1 greatly accelerates the process of ErbB-2-mediated mammary tumorigenesis, resulting in increased mammary epithelial cell proliferation and post-transcriptional up-regulation of Cyclin D1 levels. Activation of Akt-1 in this context also appears to promote mammary epithelial differentiation and interfere with metastatic progression.

Identification of Transgenic Mice.

The genotypes of transgenic mice from MMTV/AktDD-7 crosses were identified by Southern blot as described previously (15). Genotypes of animals from MMTV/NDL2–5 crosses were determined by PCR using oligonucleotide primers that amplify a region of Neu, encoding the transmembrane domain and a small portion of the extracellular domain (nucleotides 1492–2116; Ref. 23), as described previously (24).

Histological Analysis.

Complete autopsies and gross and microscopic examinations of tissues were performed. Virgin animals at various ages were sacrificed, and either the lower left mammary fat pad or tumorigenic mammary gland tissue was fixed in 4% paraformaldehyde at 4°C and blocked in paraffin. Microscopic examinations were performed on 5-μm sections stained in H&E. All whole-mounted mammary glands were prepared as described previously (25). Immunohistochemical examinations of proliferation were performed on 4-μm sections using anti-Ki67 rabbit polyclonal antibodies (NCL-Ki67p, 1:5000; Novacastra Laboratories Ltd.). The detection reaction used the Vector Elite ABC kit (Vector, Burlingame, CA).

RNA Analysis.

RNA was isolated from mouse tissues by guanidinium thiocyanate extraction, followed by cesium chloride sedimentation gradient centrifugation as described previously (25), and the RNA pellet was resuspended in 100 μl of diethyl pyrocarbonate-treated water. RNase protection assays were performed using the Ambion (Austin, TX) RPAIII kit according to instructions. Plasmid templates for riboprobe synthesis used to generate the antisense Neu (NDL2) and phosphoglycerate kinase-1 riboprobes were constructed as described previously (24). The mCYC-1 multiprobe template for Cyclin D1 antisense generation was obtained from BD-PharMingen (San Diego, CA) and contains a 231-bp fragment, which protects a 202-bp fragment of Cyclin D1 and a 125-bp fragment of glyceraldehyde-3-phosphate dehydrogenase 2. Antisense was generated for the Cyclin D1 and NDL2 riboprobes with the Ambion T7 Maxiscript kit and phosphoglycerate kinase-1 probe as described previously (24). Quantitative reverse transcription-PCR analysis was performed using the Roche (Basel, Switzerland) SYBR Green 1 RNA Amplification Kit and Light Cycler. Whey-acidic protein (WAP) transcript was amplified using primers WAP-sense (ctg cca aac caa cga gga g) and -antisense (tat ctt acg agg tcg ctg gc). β-casein transcript was amplified using primers β-casein-sense (gcc ttg cca gtc ttg cta atc) and -antisense (tcg tgg gaa gga agg tgt tc).

Protein Extraction and Analysis.

Tissue from mammary gland was flash frozen in liquid nitrogen and stored at −80°C or immediately lysed. Protein lysates were prepared as described previously (25). All immunoblots and immunoprecipitations were carried out as described previously (25) with the following exceptions. Anti-hemagglutinin (HA) immunoblot analyses were performed on 50 μg of total protein lysate and subjected to anti-HA immunoblot analysis with HA-11 mouse monoclonal antibody (1:1000; Babco-CRP, Cumberland, VA). ErbB-2 was detected by a mouse monoclonal antibody (AB-3; Oncogene Research Products). ErbB-3 (C-17, 1:500) and Grb2 (C-23, 1:1000) were detected by rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Retinoblastoma (Rb), glycogen synthase kinase-3β, 4e-bp1, and all phosphorylation analysis of these proteins was carried out on 50 μg of total lysate using polyclonal rabbit antibodies (1:1000; New England Biolabs, Beverly, MA).

Coexpression of Activated Akt-1 and ErbB-2 Results in Dramatically Faster Mammary Tumor Formation.

To examine whether Akt-1 signaling plays an important role in mammary tumor progression in the ErbB-2 model, we derived transgenic mice that coexpress both activated ErbB-2 and Akt-1 in the mammary epithelium. To accomplish this, transgenic mice carrying an oncogenic version of ErbB-2 under the MMTV promoter (MMTV/NDL2–5 strain; Ref. 20) were interbred with separate strains of mice expressing activated HA-tagged Akt-1 protein from the same mammary targeted promoter (MMTV/AktDD-7 strain; Ref. 15). The NDL2 ErbB-2 variant possesses mutations in its extracellular domain, which result in its constitutive dimerization and activation (26). The HA-tagged AktDD transgene product possesses aspartate mutations at threonine 308 and serine 473, which mimic the phosphorylated active state of the protein (27).

Previous studies have shown that the NDL2–5 strain develops multifocal metastatic mammary tumors at ∼6 months of age (20), whereas the AktDD-7 mice fail to develop mammary tumors (15). To determine the effect of Akt-1 activation on tumor formation in the NDL2–5 strain, cohorts of virgin female transgenics were monitored for tumor formation by physical palpation. The results reveal that bitransgenics show significantly reduced latency of palpable mammary tumor formation (Fig. 1,A) with 50% of the animals showing tumor formation at 104 days (n = 32) as opposed to 185 days (n = 22) in the MMTV/NDL2–5 animals alone (Fig. 1 A). As expected, the MMTV/AktDD-7 female mice failed to develop mammary tumors during the observation period.

Consistent with these kinetic analyses, whole-mount analyses of virgin mammary glands revealed that these bitransgenic animals form lesions as early as 10 weeks of age (Fig. 2), which rapidly progress to end point (∼20% body weight) in ∼1 month. To observe and compare tumor progression in the two strains of mice, mammary gland samples were taken from both strains at both 8–10 weeks of age and at end point (tumor weight > 20% body weight). On gross examination of bitransgenic mammary glands at both 8–10 weeks and end point, the lesions appear to be cyst like, consisting of a thin layer of tissue surrounding a large amount of liquid. Indeed, histological analyses of bitransgenic mammary tissue reveal the presence of atypical cystic hyperplasias with numerous foci of large cysts. These cysts are lined by irregular multilayered atypical epithelium that is papillary in some areas, although it remains within the basement membranes of the glands (Figs. 2 and 3). The cysts are similar to the tumors of MMTV/NDL2–5 animals in that the cysts are lined by papillary epithelium with typical ErbB-2 intermediate cells.

Interestingly, seven of nine of the bitransgenic lesions examined at >30 days post-tumor palpation (end point) were more differentiated (Fig. 3,D) than those of NDL2–5 animals at end point (Fig. 3,B). Indeed, the lining epithelium of the cysts of end point bitransgenics contained many gland-like spaces at both early and late stages of tumor development (Fig. 3, C and D) as compared with the NDL2–5 strain at similar stages of tumor development (Fig. 3, A and B). Consistent with this observation, of seven 8–12 week old bitransgenic mammary glands examined, all contained an unusually high number of lobuloalveolar structures not producing milk (Fig. 2 F). These observations suggest that coexpression of activated Akt-1 and ErbB-2 in the mammary epithelium can induce a differentiated phenotype at both the early and late stages of tumor development.

Significantly, unlike those of end point MMTV/NDL2–5 tumors (Fig. 3,B), the margins of the end point late stage bitransgenic lesions are pushing, and the lesions thus do not appear to be invasive (Fig. 3,D). Consistent with this observation, despite the dramatic acceleration of mammary tumorigenesis observed in the bitransgenic strain, lung tissue from these animals rarely exhibited signs of lung metastases (1 of 14 or 7%) >30 days post-tumor palpation (end point). These observations stand in direct contrast to those for female mice expressing activated ErbB-2 alone, where 8 of 12 (68%) animals had lung metastases at end point (Fig. 1 B). These observations suggest that although expression of activated Akt-1 can dramatically accelerate the induction of ErbB-2 mammary tumors, the tumors formed are inherently nonmetastatic.

Coexpression of NDL2–5 and HA-AktDD Leads to Enhanced ErbB-3 Levels at an Earlier Age than in the NDL2–5 Strain Alone.

To confirm that the accelerated tumor phenotype observed in bitransgenic mice correlated with coexpression of the AktDD and ErbB-2 gene products, we examined NDL transcript levels and performed immunoblot analyses against ErbB-2 and the HA-tagged AktDD on mammary gland samples from the various mouse strains at both 8–10 weeks of age and end point. Expression of HA-tagged AktDD could be readily detected in all AktDD-7 and NDL2–5XAktDD-7 mammary tissues (Fig. 4,A, Lanes 2–3 and 8–11) but not in negative control samples from either FVB or NDL2–5 mice (Fig. 4,A, Lanes 1, 4–7). Consistent with previous studies (20, 28), ErbB-2 protein levels were quite low in 8–10-week-old NDL2–5 mammary gland samples, although the NDL transcript is easily detected (Fig. 4, A, Lanes 4–5 and B, Lanes 4–5). As in previous studies (20, 28), we observed elevated levels of ErbB-2 in NDL2–5 mammary tumors at end point (Fig. 4,A, Lanes 6–7) relative to 8–10-week-old NDL2–5 mammary tissue. Similarly, examination of end point bitransgenic mammary tumors also revealed elevated expression of ErbB-2 (Fig. 4,A, Lanes 10–11) relative to 8–10-week-old NDL2–5 mammary tissue. Significantly, we also frequently observed up-regulation of ErbB-2 protein in bitransgenic mammary tissue at 8–10 weeks of age (Fig. 4 A, Lane 8) relative to 8–10-week-old NDL2–5 mammary tissue.

Because ErbB-3 overexpression is also frequently associated with NDL2–5 mammary tumorigenesis (20), we measured the levels of ErbB-3 in these transgenic mammary samples. Consistent with a previous study (20), levels of ErbB-3 correlated with those of ErbB-2 (Fig. 4,A). The differences in the levels of HA-AktDD, ErbB-2, and ErbB-3 were not caused by protein loading, because all tissues expressed comparable levels of Grb2 protein (Fig. 4 A).

Consistent with these observations, we have demonstrated an identical pattern of ErbB-2 and ErbB-3 expression, with 50% of samples showing elevated ErbB-2 and ErbB-3 expression, in a larger independent cohort (n = 4) of samples (data not shown). The incomplete penetrance observed for this phenotype likely reflects a wide variation in the age at which overexpression of ErbB-2 and ErbB-3 occurs during mammary tumorigenesis in the bitransgenic strain. In support of this hypothesis, tumor formation in the bitransgenic strain occurs with widely disparate latencies (Fig. 1 A). Taken together, these results demonstrate that the dramatic acceleration in tumor onset in the bitransgenic strain correlates with elevated expression of ErbB-2 and ErbB-3.

Coexpression of AktDD and ErbB-2 Is Associated with Increases in Cellular Proliferation at 8–10 Weeks of Age.

Akt-1 activation has shown previously to promote mammary tumorigenesis by inhibiting apoptosis (15). To determine whether Akt-1 activation plays a similar role in this cross, we measured apoptosis levels in the various transgenic strains by terminal deoxynucleotidyl transferase-mediated nick end labeling assay (29). These analyses revealed negligible apoptotic levels in mammary gland epithelium sampled at various ages from both the MMTV/NDL2–5 and MMTV/AktDD-7XNDL2–5 strains, which were comparable with wild-type levels (data not shown). Consistent with these results, analysis of FKHR phosphorylation failed to reveal any substantial increases in phosphorylation of serine 256 in bitransgenic mammary glands as compared with controls (data not shown).

Because Akt-1 can also regulate cell cycle progression (30, 31, 32), we examined the mammary epithelial proliferation index of the transgenic strains by anti-Ki67 immunohistochemistry at 8–10 weeks of age (Fig. 5, A and B). These analyses reveal that 8–10 week bitransgenic mammary epithelia have higher proliferation indices than age-matched MMTV/NDL2–5 mammary epithelia (Fig. 5,C). To examine the molecular basis for this increase, we assessed the status of some of the Akt-1 targets implicated in cell cycle regulation. Akt-1 has been demonstrated to down-regulate levels of the cell cycle inhibitor p27/Kip1 by phosphorylating and inhibiting the forkhead family of transcription factors (33). However, consistent with our FKHR phosphorylation results, we observed no substantial changes in p27 levels in bitransgenic mammary glands as compared with controls (Fig. 6). Akt-1 can also regulate cell proliferation through its effects on Cyclin D1 (34). Examination of mammary Cyclin D1 protein levels revealed that Cyclin D1 is overexpressed in a significant proportion of bitransgenic mammary glands at 8–10 weeks of age at similar levels to that observed in end point MMTV/NDL2–5 and bitransgenic mammary tumors (Fig. 6, Lanes 6–11). Significantly, these increases in Cyclin D1 levels are not observed in MMTV/NDL2–5 mammary glands at 8–10 weeks of age (Fig. 6, compare Lanes 4–5 with 8–9). Consistent with these observations, we have demonstrated an identical pattern of Cyclin D1 overexpression in a second independent cohort of samples (data not shown).

As Cyclin D1 promotes cell cycle progression via its ability to promote phosphorylation of Rb and allow exit from G1(35), we examined the phosphorylation status of Rb by immunoblot analysis with Rb phosphospecific antibodies. These analyses revealed slightly increased Rb phosphorylation relative to wild type in most mammary epithelial samples expressing increased levels of Cyclin D1 at 8–10 weeks and end point in both the NDL2–5 and bitransgenic strains (Fig. 6, Lanes 6–11).

To explore the molecular basis of the up-regulation of Cyclin D1 in these samples, we measured the levels of Cyclin D1 transcript. These analyses reveal that Cyclin D1 transcript levels are comparable in mammary gland samples expressing either high or low levels of Cyclin D1 protein (Fig. 7, A and B, compare Lanes 6–12 with 4–5). Consistent with previous results (15), these observations suggest that Akt-1 acts to increase Cyclin D1 protein levels via a post-transcriptional mechanism.

Coexpression of Akt-1 and ErbB-2 Results in the Induction of Highly Differentiated Tumors.

To further examine the enhanced differentiation observed in end point bitransgenic mammary lesions, we performed quantitative reverse transcription-PCR for transcripts of the mammary epithelial differentiation markers WAP and β-casein on samples derived from both NDL2–5 and bitransgenic mammary tumor tissues at end point. All bitransgenic samples were derived from mice exhibiting enhanced mammary differentiation as determined pathologically. Additionally, none of these mice developed metastases >30 days after mammary tumor palpation. Of the NDL2–5 controls examined, 60% had developed metastases at end point. Consistent with our pathological observations, the results of these reverse transcription-PCR analyses revealed substantially higher levels of both WAP and β-casein transcripts in end point bitransgenic mammary tumors (Fig. 8). Taken together, these results further support the suggestion that expression of activated Akt-1 can promote the differentiation of mammary epithelium expressing activated ErbB-2.

In this study, we examined the consequences of mammary epithelial activation of Akt-1 on ErbB-2 (NDL2)-mediated mammary tumorigenesis by generating bitransgenics expressing activated Akt-1 and ErbB-2 transgenes. The results of our studies reveal that coexpression of the activated Akt-1 and ErbB-2 transgenes results in the formation of mammary lesions with shorter latency than in animals expressing activated ErbB-2 alone (Fig. 1). The rapid induction of mammary tumors as early as 8–10 weeks of age in the bitransgenic strain correlated with overexpression of ErbB-2 and ErbB-3, elevated Cyclin D1 protein levels, and dramatic increases in the mammary epithelial proliferative index (Figs. 4,5,6,7). However, in contrast to the metastatic tumors induced in the parental NDL2–5 strain, tumors coexpressing Akt-1 and ErbB-2 were poorly invasive and rarely metastasized (Figs. 1 and 3). The poor metastatic behavior of the bitransgenic tumors correlated with differentiated pathology at end point (Figs. 3 and 8).

Given the correlation between elevated levels of ErbB-2, ErbB-3, and mammary tumor formation in various activated ErbB-2 transgenic strains observed previously, our results showing early up-regulation of ErbB-3 and ErbB-2 in the bitransgenics relative to the NDL2–5 strain suggest that Akt-1 activation may be acting in this cross to up-regulate the molecular pathways involved in ErbB-2-mediated mammary tumorigenesis. Importantly, our results do not preclude the possibility that the endogenous Akt-1 contributes to some degree to tumorigenesis in the bitransgenic strain. Indeed, the increased levels of ErbB-3, a known activator of Akt-1 (22), in the bitransgenic and strain relative to 8–10-week-old NDL2–5 and wild-type samples support this possibility.

The observation that activated Akt-1 can cooperate with activated ErbB-2 is consistent with previous studies showing that activated Akt-1 can accelerate mammary tumor induction in transgenic mice expressing a mutant polyomavirus middle T antigen oncogene decoupled from the PI-3K signaling pathway (15). Our observations suggest that Akt-1 may contribute to accelerated tumorigenesis in the bitransgenics through its effects on cell cycle progression and cellular proliferation. To further explore this observation we examined the status of cell cycle regulatory proteins known to be regulated by Akt-1. Consistent with results from our previous transgenic cross (15), coexpression of activated Akt-1 and ErbB-2 has little effect on overall p27 levels. However, because Akt-1 can also prevent G1 arrest by inhibiting p27 nuclear import (30, 32, 36), our results do not exclude the possibility that Akt-1 activation affects cell cycle progression by modulating p27 localization in bitransgenic mammary epithelial cells.

Another protein known to regulate cell cycle progression in cancer progression is Cyclin D1. Indeed, Cyclin D1 is up-regulated in ∼50% of mammary tumors (37) and is essential for ErbB-2-mediated tumor progression (38). Furthermore, overexpression of Cyclin D1 under control of the MMTV promoter results in the development of focal mammary tumors (39). Consistent with these studies, we observe elevated levels of Cyclin D1 protein in mammary tissues of 8–10-week-old animals coexpressing activated ErbB-2 and Akt-1 as compared with NDL2–5 mammary gland samples of the same age (Fig. 5). Mammary gland samples expressing elevated levels of Cyclin D1 also generally exhibit slightly increased Rb phosphorylation relative to NDL2–5 mammary samples at 8–10 weeks, suggesting that the increased Cyclin D1 protein levels affect cell cycle regulation (Fig. 5). Interestingly, mammary gland Cyclin D1 transcript levels do not increase alongside Cyclin D1 protein levels, suggesting that Cyclin D1 protein levels are regulated at a post-transcriptional level. Although our results do not eliminate an indirect mechanism of Akt-1-induced Cyclin D1 protein overexpression, PI-3K/Akt-1 signaling is known to regulate distinct pathways involved in post-transcriptional regulation of Cyclin D1 levels, e.g., Akt-1 phosphorylates and inactivates glycogen synthase kinase-3β (40), which can target Cyclin D1 for ubiquitin-mediated proteasomal degradation (41). Akt-1 signaling might also affect Cyclin D1 levels through regulation of translation initiation, because Akt-1 regulates the activity of members of the rapamycin-sensitive translation initiation complex (42, 43) shown to regulate Cyclin D1 protein levels (44). Ultimately, additional studies will be necessary to investigate the mechanism responsible for the post-transcriptional increase in Cyclin D1 levels. However, overall, these results suggest that Akt-1 activation can accelerate the early stages of ErbB-2-mediated tumorigenesis through increased Cyclin D1 expression and cellular proliferation. As such, these studies further support a central role for Cyclin D1 in ErbB-2-mediated mammary tumorigenesis. However, given the correlative nature of these results, additional studies using bitransgenic strategies to examine a putative causative role for Cyclin D1 in ErbB-2 tumorigenesis are necessary to further strengthen this assertion.

Despite the dramatic acceleration of tumor induction in mice coexpressing activated Akt-1 and ErbB-2, the bitransgenic tumors were poorly invasive and rarely metastasized (Fig. 1). In contrast, activated ErbB-2 tumors are highly invasive and frequently metastasized to the lungs (Fig. 1; Ref. 20). These results would predict longer survival times for bitransgenic as compared with NDL2–5 animals, because bitransgenic tumors are less aggressive. However, as noted previously, the maximum allowable end point in our studies is defined as the point when tumor load reaches ≤20% body weight, so a comparison of survival time was not workable. However, an approximate comparison of tumor aggressiveness using length of time to sacrifice and tumor characteristics is possible. In this respect, although both bitransgenics and NDL2–5 transgenics typically progress to end point ∼1 month post-tumor palpation, NDL2–5 mammary tumors at end point are solid, whereas the tumors in the bitransgenic strain are cyst like and fluid filled. Given that these bitransgenics show negligible levels of lung metastases, and it is mainly this increase of fluid in the bitransgenic tumors which increases tumor size and weight, these results suggest that the bitrangenics would survive longer than the NDL2–5 transgenics, if only tumor aggressiveness and metastasis were taken into account.

It is unclear at this time which stage of ErbB-2-mediated metastasis Akt-1 activation suppresses. Mammary epithelial cells undergo multiple functional changes during the transition between the tumor development and metastatic stages of breast cancer. These changes encompass modifications in their cell–cell and cell–matrix adhesions, along with increases in their anoikis resistance, motility, protease secretion, extracellular matrix, and invasiveness (45). Given the extensive evidence demonstrating prosurvival roles for Akt-1 (46), it seems unlikely that the bitransgenic mammary epithelial cells in our study are more prone to anoikis. Importantly, although it has been demonstrated that Akt-1 does not promote ErbB-2-mediated motility, it has not been shown that Akt-1 inhibits this process (47).

Interestingly, the dramatic inhibition of tumor metastasis was associated with increases in the differentiation status of the mammary tumors. Proliferation and differentiation are classically thought of as two separate and opposing processes with differentiated cells exiting the cell cycle (48). However, in basic terms, differentiated cells are cells which have undergone morphogenesis to perform a specific function. The mammary gland is unique in that it can undergo multiple cycles of differentiation and subsequent loss of this functional status (49). Differentiation of the mammary epithelium during pregnancy includes ductal outgrowth, the formation of specialized lobuloalveolar structures, and the secretion of milk proteins (49). Thus, in the mammary gland, pregnancy-associated differentiation is associated with mammary epithelial cellular proliferation.

Multiple studies have demonstrated a negative correlation between tumor differentiation and metastasis (50, 51). The insulin-like growth factor (IGF) pathway strongly activates Akt-1 signaling (27), and IGF1-receptor is frequently overexpressed in differentiated breast tumors (52). Similarly, consistent with our results showing reduction of metastasis associating with Akt-1 activation, reduced expression of IGF-1 leads to a more metastatic phenotype in MCF-7 cells (53). The cellular basis of this relationship between metastasis and differentiation is unclear, but the specialized functions of differentiated mammary epithelial cells, such as milk production, rely on several characteristics of the cellular environment, including the presence of specific extracellular matrix and cell–cell adhesions (54). Significantly, Akt-1 positively regulates the production of laminin and collagen isotypes of the basement membrane (55), which are implicated in the control of mammary epithelial cell differentiation (56). Thus, the enhanced differentiation observed in the bitransgenic tumors may be a result of Akt-1 up-regulation of laminin and collagen production. This hypothesis also suggests that Akt-1 may hinder metastasis in the bitransgenics by preventing the degradation of the extracellular matrix. Additional studies will be necessary to investigate this intriguing possibility and determine the exact effect of Akt-1 activation on the metastatic properties of activated ErbB-2-overexpressing mammary epithelial cells. Taken together, these preliminary results suggest that Akt-1 activation may suppress ErbB-2-mediated mammary tumor invasion by promoting differentiation of the mammary epithelium. Further elucidation of the molecular mechanism by which Akt-1 suppresses metastatic progression may provide insight into potential avenues to therapeutically interfere with metastatic progression.

Our results would further suggest that Akt-1 activation plays a role in normal mammary gland differentiation. In this regard, it has been reported that mammary-specific ablation of PTEN results in precocious mammary gland differentiation, specifically lobuloalveolar development (57). Conversely, elevated expression of PTEN suppresses mouse mammary epithelial differentiation (58). Furthermore, treatment of the mammary gland with components of the IGF/IGF receptor axis that strongly activate Akt-1 induces precocious mammary differentiation (59).

Taken together, these observations suggest that coexpression of activated Akt-1 with activated ErbB-2 can accelerate the early stages of ErbB-2-mediated tumorigenesis through increased cellular proliferation but interferes with subsequent metastatic progression by inducing mammary epithelial differentiation.

Grant support: Grants awarded to W. Muller include NIH PPG CA099031 and Canadian Breast Cancer Research Initiative. National Cancer Institute of Canada and Medical Research Council Grants awarded to J. Woodgett. W. Muller is a recipient of a Medical Research Council of Canada Scientist award, J. Woodgett is a recipient of a Medical Research Council Senior Scientist award, and J. Hutchinson was supported by a scholarship from the United States Army Medical Research’s Breast Cancer Research Program.

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.

Requests for reprints: William J. Muller, Molecular Oncology Group, McGill University, Royal Victoria Hospital–Room H5.21, Montreal, Quebec, H3A 1A1, Canada.

Fig. 1.

Coexpression of HA-AktDD and NDL2 results in decreased latency of mammary tumor formation. A, mammary tumor kinetics of mouse mammary tumor virus/NDL2–5 and mouse mammary tumor virus/NDL2–5XAktDD-7strains. Age indicated is that at which a mammary tumor is first palpable. The number of animals analyzed for each strain (n) and median age (T50) at which mammary tumors are palpable are also shown. B, percentage of mammary tumor-bearing animals with metastatic lesions in the lung >30 days after mammary tumor palpation (end point). The percentage of tumor-bearing mice harboring lung metastases and total number of animals analyzed (n) are indicated for each genotype. A Fisher exact test demonstrates a significant difference between occurrence of metastases in NDL2–5 and NDL2–5XAktDD-7 mice (P = 0.002).

Fig. 1.

Coexpression of HA-AktDD and NDL2 results in decreased latency of mammary tumor formation. A, mammary tumor kinetics of mouse mammary tumor virus/NDL2–5 and mouse mammary tumor virus/NDL2–5XAktDD-7strains. Age indicated is that at which a mammary tumor is first palpable. The number of animals analyzed for each strain (n) and median age (T50) at which mammary tumors are palpable are also shown. B, percentage of mammary tumor-bearing animals with metastatic lesions in the lung >30 days after mammary tumor palpation (end point). The percentage of tumor-bearing mice harboring lung metastases and total number of animals analyzed (n) are indicated for each genotype. A Fisher exact test demonstrates a significant difference between occurrence of metastases in NDL2–5 and NDL2–5XAktDD-7 mice (P = 0.002).

Close modal
Fig. 2.

Coexpression of HA-AktDD and NDL2 results in faster focal mammary lesion formation than expression of NDL2 alone. Black and white panel showing whole mounts (A, C, and E) and histology (B, D, and F) of mammary glands from 12-week-old nulliparous normal wild-type FVB (A and B), NDL2–5 (C and D), and NDL2–5XAktDD-7 (E and F) mice. Note the increased side branching in the NDL2–5 gland without focal lesions (C and D) and numerous focal lesions in the bitransgenic gland (E and F).

Fig. 2.

Coexpression of HA-AktDD and NDL2 results in faster focal mammary lesion formation than expression of NDL2 alone. Black and white panel showing whole mounts (A, C, and E) and histology (B, D, and F) of mammary glands from 12-week-old nulliparous normal wild-type FVB (A and B), NDL2–5 (C and D), and NDL2–5XAktDD-7 (E and F) mice. Note the increased side branching in the NDL2–5 gland without focal lesions (C and D) and numerous focal lesions in the bitransgenic gland (E and F).

Close modal
Fig. 3.

Coexpression of HA-AktDD and NDL2 results in the formation of differentiated noninvasive lesions. Black and white image showing the small (early stage of growth) but solid lesions (A) and larger solid lesions (late stage of growth; B) from a NDL2–5 female at 40 weeks of age (end point). Compare these lesions with the more glandular and subdivided small (early stage; C) and large (late stage; D) lesions from an NDL2–5XAktDD-7 mouse at 19 and 32 (end point) weeks of age, respectively. Note the relative organization of the lesions and solid versus small cluster growth.

Fig. 3.

Coexpression of HA-AktDD and NDL2 results in the formation of differentiated noninvasive lesions. Black and white image showing the small (early stage of growth) but solid lesions (A) and larger solid lesions (late stage of growth; B) from a NDL2–5 female at 40 weeks of age (end point). Compare these lesions with the more glandular and subdivided small (early stage; C) and large (late stage; D) lesions from an NDL2–5XAktDD-7 mouse at 19 and 32 (end point) weeks of age, respectively. Note the relative organization of the lesions and solid versus small cluster growth.

Close modal
Fig. 4.

Mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands overexpress ErbB-3 at an earlier age than NDL2–5 mammary glands. A, immunoblot analyses of expression of HA-AktDD, ErbB-2, ErbB-3, and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 40–42-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–24-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of HA-AktDD (top panel), ErbB-2 (top middle panel), ErbB-3 (bottom middle panel), and a Grb2 loading control (bottom panel). Numbers above each lane, mouse identification numbers. B, RNase protection assay analysis of expression of NDL2 and PGK-1 (loading control) in mammary glands from matched samples to A. Arrows, the migration of the protected fragments of NDL2 (top panel) and PGK-1 (bottom panel). Numbers above each lane, mouse identification numbers.

Fig. 4.

Mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands overexpress ErbB-3 at an earlier age than NDL2–5 mammary glands. A, immunoblot analyses of expression of HA-AktDD, ErbB-2, ErbB-3, and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 40–42-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–24-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of HA-AktDD (top panel), ErbB-2 (top middle panel), ErbB-3 (bottom middle panel), and a Grb2 loading control (bottom panel). Numbers above each lane, mouse identification numbers. B, RNase protection assay analysis of expression of NDL2 and PGK-1 (loading control) in mammary glands from matched samples to A. Arrows, the migration of the protected fragments of NDL2 (top panel) and PGK-1 (bottom panel). Numbers above each lane, mouse identification numbers.

Close modal
Fig. 5.

Coexpression of NDL2 and HA-AktDD increases the proliferation index of mammary epithelial cells at an earlier age than NDL2 alone. Anti-Ki67 immunohistochemical analysis of virgin mammary glands from mouse mammary tumor virus/NDL2–5 (A) and mouse mammary tumor virus/NDL2–5XAktDD-7 (B) strains at 10 weeks of age. Cells were stained brown for Ki67 with 3,3′-diaminobenzidine and counterstained with hematoxylin. Note the increased staining in the mouse mammary tumor virus/NDL2–5XAktDD-7 sample (B) as compared with the age-matched mouse mammary tumor virus/NDL2–5 sample (A). C, mammary epithelial proliferation indices of mouse mammary tumor virus/NDL2–5 and mouse mammary tumor virus/AktDD-7XNDL2–5 strains at 8–10 weeks of age. Values shown represent the percentage of total mammary epithelial cells stained positive for apoptosis by anti-Ki67 immunohistochemistry in virgin female mice at 8–10 weeks of age. Five randomly chosen fields with a minimum of 150 cells were counted in four mice from each strain. Numbers shown on graph, the mean, 95% confidence interval, and number of mice examined (n). Difference between averages is statistically significant as determined by one-tailed heteroscedastic Student’s t test (P < 0.05).

Fig. 5.

Coexpression of NDL2 and HA-AktDD increases the proliferation index of mammary epithelial cells at an earlier age than NDL2 alone. Anti-Ki67 immunohistochemical analysis of virgin mammary glands from mouse mammary tumor virus/NDL2–5 (A) and mouse mammary tumor virus/NDL2–5XAktDD-7 (B) strains at 10 weeks of age. Cells were stained brown for Ki67 with 3,3′-diaminobenzidine and counterstained with hematoxylin. Note the increased staining in the mouse mammary tumor virus/NDL2–5XAktDD-7 sample (B) as compared with the age-matched mouse mammary tumor virus/NDL2–5 sample (A). C, mammary epithelial proliferation indices of mouse mammary tumor virus/NDL2–5 and mouse mammary tumor virus/AktDD-7XNDL2–5 strains at 8–10 weeks of age. Values shown represent the percentage of total mammary epithelial cells stained positive for apoptosis by anti-Ki67 immunohistochemistry in virgin female mice at 8–10 weeks of age. Five randomly chosen fields with a minimum of 150 cells were counted in four mice from each strain. Numbers shown on graph, the mean, 95% confidence interval, and number of mice examined (n). Difference between averages is statistically significant as determined by one-tailed heteroscedastic Student’s t test (P < 0.05).

Close modal
Fig. 6.

Overexpression of Cyclin D1 correlates with increased phosphorylation of retinoblastoma (Rb) in 8–10-week-old mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands. Immunoblot analyses of expression of Cyclin D1, p27, Phospho-Rb(Ser780), Rb, and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 33- to 40-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–32-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of Cyclin D1 (top panel), p27 (second highest panel), Phospho-Rb(Ser780; middle panel), Rb (second lowest panel), and Grb2 loading control (lowest panel). Numbers above each lane, mouse identification numbers. Note the increase in Cyclin D1 protein levels and general increase in Rb phosphorylation in the 8–10-week-old mouse mammary tumor virus/NDL2–5XAktDD-7 samples (Lanes 8 and 9) as compared with the age-matched mouse mammary tumor virus/NDL2–5 samples (Lanes 4 and 5).

Fig. 6.

Overexpression of Cyclin D1 correlates with increased phosphorylation of retinoblastoma (Rb) in 8–10-week-old mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands. Immunoblot analyses of expression of Cyclin D1, p27, Phospho-Rb(Ser780), Rb, and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 33- to 40-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–32-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of Cyclin D1 (top panel), p27 (second highest panel), Phospho-Rb(Ser780; middle panel), Rb (second lowest panel), and Grb2 loading control (lowest panel). Numbers above each lane, mouse identification numbers. Note the increase in Cyclin D1 protein levels and general increase in Rb phosphorylation in the 8–10-week-old mouse mammary tumor virus/NDL2–5XAktDD-7 samples (Lanes 8 and 9) as compared with the age-matched mouse mammary tumor virus/NDL2–5 samples (Lanes 4 and 5).

Close modal
Fig. 7.

Cyclin D1 is post-transcriptionally overexpressed in mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands. A, immunoblot analyses of expression of Cyclin D1 and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 33- to 40-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–32-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of Cyclin D1 (top panel) and Grb2 loading control (bottom panel). Numbers above each lane, mouse identification numbers. B, RNase protection assay analysis of Cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2 (loading control) expression in mammary glands from matched samples to A. Arrows, the migration of the protected fragments of Cyclin D1 (top panel) and GAPDH2 (bottom panel). Numbers above each lane, mouse identification numbers.

Fig. 7.

Cyclin D1 is post-transcriptionally overexpressed in mouse mammary tumor virus/NDL2–5XAktDD-7 mammary glands. A, immunoblot analyses of expression of Cyclin D1 and Grb2 in mammary glands from 8- to 10-week-old virgin females (8–10wk) from FVB (Lane 1), mouse mammary tumor virus/AktDD-7 (Lanes 2 and 3), mouse mammary tumor virus/NDL2–5 (Lanes 4 and 5), mouse mammary tumor virus/NDL2–5XAktDD-7 (Lanes 8 and 9), and mammary tumors (MT) from 33- to 40-week-old (end point) mouse mammary tumor virus/NDL2–5 virgin females (Lanes 6 and 7) and 23–32-week-old (end point) virgin mouse mammary tumor virus/NDL2–5XAktDD-7 females (Lanes 10 and 11). Arrows, the migration of Cyclin D1 (top panel) and Grb2 loading control (bottom panel). Numbers above each lane, mouse identification numbers. B, RNase protection assay analysis of Cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2 (loading control) expression in mammary glands from matched samples to A. Arrows, the migration of the protected fragments of Cyclin D1 (top panel) and GAPDH2 (bottom panel). Numbers above each lane, mouse identification numbers.

Close modal
Fig. 8.

Mouse mammary tumor virus/NDL2–5XAktDD-7 mammary tumors at end point express higher levels of differentiation marker transcripts than NDL2–5 mammary tumors. Quantitative reverse transcription-PCR analysis of β-casein (A) and whey-acidic protein (WAP; B) transcript expression. Mammary tumor samples from six virgin female of each strain at end point were analyzed in triplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels, and the values were averaged for each transgenic strain. All bitransgenic samples showed differentiated pathology and were derived from animals not exhibiting metastasis >30 days after mammary tumor palpation. A majority (60%) of the NDL2–5 samples was derived from animals exhibiting metastasis to the lung at a similar stage. Numbers shown on graph, the mean, 95% confidence interval, and number of mice examined (n). Difference between averages for both graphs is statistically significant as determined by one-tailed heteroscedastic Student’s t test (P < 0.05).

Fig. 8.

Mouse mammary tumor virus/NDL2–5XAktDD-7 mammary tumors at end point express higher levels of differentiation marker transcripts than NDL2–5 mammary tumors. Quantitative reverse transcription-PCR analysis of β-casein (A) and whey-acidic protein (WAP; B) transcript expression. Mammary tumor samples from six virgin female of each strain at end point were analyzed in triplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels, and the values were averaged for each transgenic strain. All bitransgenic samples showed differentiated pathology and were derived from animals not exhibiting metastasis >30 days after mammary tumor palpation. A majority (60%) of the NDL2–5 samples was derived from animals exhibiting metastasis to the lung at a similar stage. Numbers shown on graph, the mean, 95% confidence interval, and number of mice examined (n). Difference between averages for both graphs is statistically significant as determined by one-tailed heteroscedastic Student’s t test (P < 0.05).

Close modal

We thank Dinsdale Gooden and Alison Gordon for oligonucleotide synthesis (MOBIX Central Facility, McMaster University). We also thank Monica Graham, Carrie Merola, and Judy Walls for technical support. Finally, we thank Robert Munn for generating the images used in Figs. 2 and 3.

1
Dankort DL, Muller WJ Signal transduction in mammary tumorigenesis: a transgenic perspective.
Oncogene
,
19
:
1038
-44,  
2000
.
2
Slamon DJ, Godolphin W, Jones LA, et al Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science
,
244
:
707
-12,  
1989
.
3
Mansour EG, Ravdin PM, Dressler L Prognostic factors in early breast carcinoma.
Cancer
,
74
:
381
-400,  
1994
.
4
Andrulis IL, Bull SB, Blackstein ME, et al neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group.
J Clin Oncol
,
16
:
1340
-9,  
1998
.
5
Osborne CK, Bardou V, Hopp TA, et al Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer.
J Natl Cancer Inst (Bethesda)
,
95
:
353
-61,  
2003
.
6
Zhou BP, Hu MC, Miller SA, et al HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-κB pathway.
J Biol Chem
,
275
:
8027
-31,  
2000
.
7
Fruman DA, Meyers RE, Cantley LC Phosphoinositide kinases.
Annu Rev Biochem
,
67
:
481
-507,  
1998
.
8
Chan TO, Rittenhouse SE, Tsichlis PN AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation.
Annu Rev Biochem
,
68
:
965
-1014,  
1999
.
9
Vivanco I, Sawyers CL The phosphatidylinositol 3-Kinase AKT pathway in human cancer.
Nat Rev Cancer
,
2
:
489
-501,  
2002
.
10
Staal SP Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma.
Proc Natl Acad Sci USA
,
84
:
5034
-7,  
1987
.
11
Perez-Tenorio G, Stal O Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients.
Br J Cancer
,
86
:
540
-5,  
2002
.
12
Sun M, Wang G, Paciga JE, et al AKT1/PKBα kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.
Am J Pathol
,
159
:
431
-7,  
2001
.
13
Stal O, Perez-Tenorio G, Akerberg L, et al Akt kinases in breast cancer and the results of adjuvant therapy.
Breast Cancer Res
,
5
:
R37
-44,  
2003
.
14
Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway.
Proc Natl Acad Sci USA
,
95
:
15587
-91,  
1998
.
15
Hutchinson J, Jin J, Cardiff RD, Woodgett JR, Muller WJ Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression.
Mol Cell Biol
,
21
:
2203
-12,  
2001
.
16
Schwertfeger KL, Richert MM, Anderson SM Mammary gland involution is delayed by activated Akt in transgenic mice.
Mol Endocrinol
,
15
:
867
-81,  
2001
.
17
Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.
Nat Cell Biol
,
3
:
973
-82,  
2001
.
18
Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.
Mol Cell Biol
,
21
:
3995
-4004,  
2001
.
19
Lenferink AE, Busse D, Flanagan WM, Yakes FM, Arteaga CL ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways.
Cancer Res
,
61
:
6583
-91,  
2001
.
20
Siegel PM, Ryan ED, Cardiff RD, Muller WJ Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer.
EMBO J
,
18
:
2149
-64,  
1999
.
21
Pawson T Protein modules and signalling networks.
Nature
,
373
:
573
-80,  
1995
.
22
Hellyer NJ, Kim MS, Koland JG Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.
J Biol Chem
,
276
:
42153
-61,  
2001
.
23
Bargmann CI, Hung MC, Weinberg RA Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185.
Cell
,
45
:
649
-57,  
1986
.
24
Siegel PM, Dankort DL, Hardy WR, Muller WJ Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.
Mol Cell Biol
,
14
:
7068
-77,  
1994
.
25
Webster MA, Hutchinson JN, Rauh MJ, et al Requirement for both Shc and phosphatidylinositol 3′ kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis.
Mol Cell Biol
,
18
:
2344
-59,  
1998
.
26
Siegel PM, Muller WJ Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.
Proc Natl Acad Sci USA
,
93
:
8878
-83,  
1996
.
27
Alessi DR, Andjelkovic M, Caudwell B, et al Mechanism of activation of protein kinase B by insulin and IGF-1.
EMBO J
,
15
:
6541
-51,  
1996
.
28
Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease.
Proc Natl Acad Sci USA
,
89
:
10578
-82,  
1992
.
29
Gavrieli Y, Sherman Y, Ben-Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol
,
119
:
493
-501,  
1992
.
30
Liang J, Zubovitz J, Petrocelli T, et al PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest.
Nat Med
,
8
:
1153
-60,  
2002
.
31
Rossig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells.
Mol Cell Biol
,
21
:
5644
-57,  
2001
.
32
Shin I, Yakes FM, Rojo F, et al PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization.
Nat Med
,
8
:
1145
-52,  
2002
.
33
Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, Sellers WR Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN.
Mol Cell Biol
,
20
:
8969
-82,  
2000
.
34
Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway.
J Biol Chem
,
273
:
29864
-72,  
1998
.
35
Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.
Genes Dev
,
7
:
331
-42,  
1993
.
36
Viglietto G, Motti ML, Bruni P, et al Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer.
Nat Med
,
8
:
1136
-44,  
2002
.
37
Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J Cyclin D1 protein expression and function in human breast cancer.
Int J Cancer
,
57
:
353
-61,  
1994
.
38
Yu Q, Geng Y, Sicinski P Specific protection against breast cancers by cyclin D1 ablation.
Nature
,
411
:
1017
-21,  
2001
.
39
Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice.
Nature
,
369
:
669
-71,  
1994
.
40
van Weeren PC, de Bruyn KM, de Vries-Smits AM, van Lint J, Burgering BM Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB.
J Biol Chem
,
273
:
13150
-6,  
1998
.
41
Diehl JA, Cheng M, Roussel MF, Sherr CJ Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization.
Genes Dev
,
12
:
3499
-511,  
1998
.
42
Dufner A, Andjelkovic M, Burgering BM, Hemmings BA, Thomas G Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation.
Mol Cell Biol
,
19
:
4525
-34,  
1999
.
43
Gingras AC, Kennedy SG, O’Leary MA, Sonenberg N, Hay N 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway.
Genes Dev
,
12
:
502
-13,  
1998
.
44
Grewe M, Gansauge F, Schmid RM, Adler G, Seufferlein T Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells.
Cancer Res
,
59
:
3581
-7,  
1999
.
45
Eccles SA The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis.
J Mammary Gland Biol Neoplasia
,
6
:
393
-406,  
2001
.
46
Datta SR, Brunet A, Greenberg ME Cellular survival: a play in three Akts.
Genes Dev
,
13
:
2905
-27,  
1999
.
47
Woods Ignatoski KM, Livant DL, Markwart S, Grewal NK, Ethier SP The Role of phosphatidylinositol 3′-kinase and its downstream signals in erbB-2-mediated transformation.
Mol Cancer Res
,
1
:
551
-60,  
2003
.
48
Blagosklonny MV Apoptosis, proliferation, differentiation: in search of the order.
Semin Cancer Biol
,
13
:
97
-105,  
2003
.
49
Hennighausen L, Robinson GW Think globally, act locally: the making of a mouse mammary gland.
Genes Dev
,
12
:
449
-55,  
1998
.
50
Herrera-Gayol A, Royal A, Babai F Correlation between cell differentiation stage, types of invasion, and hematogenous metastasis in experimental rhabdomyosarcomas.
Exp Mol Pathol
,
63
:
1
-15,  
1995
.
51
Eccles SA Differentiation and neoplasia. Invasion and metastasis; experimental systems.
J Pathol
,
141
:
333
-53,  
1983
.
52
Papa V, Gliozzo B, Clark GM, et al Insulin-like growth factor-I receptors are overexpressed and predict a low risk in human breast cancer.
Cancer Res
,
53
:
3736
-40,  
1993
.
53
Pennisi PA, Barr V, Nunez NP, Stannard B, Le Roith D Reduced expression of insulin-like growth factor I receptors in MCF-7 breast cancer cells leads to a more metastatic phenotype.
Cancer Res
,
62
:
6529
-37,  
2002
.
54
Streuli C Extracellular matrix remodelling and cellular differentiation.
Curr Opin Cell Biol
,
11
:
634
-40,  
1999
.
55
Li X, Talts U, Talts JF, Arman E, Ekblom P, Lonai P Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.
Proc Natl Acad Sci USA
,
98
:
14416
-21,  
2001
.
56
Streuli CH, Bailey N, Bissell MJ Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell–cell interaction and morphological polarity.
J Cell Biol
,
115
:
1383
-95,  
1991
.
57
Li G, Robinson GW, Lesche R, et al Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland.
Development
,
129
:
4159
-70,  
2002
.
58
Dupont J, Renou JP, Shani M, Hennighausen L, LeRoith D PTEN overexpression suppresses proliferation and differentiation and enhances apoptosis of the mouse mammary epithelium.
J Clin Investig
,
110
:
815
-25,  
2002
.
59
Ruan W, Newman CB, Kleinberg DL Intact and amino-terminally shortened forms of insulin-like growth factor I induce mammary gland differentiation and development.
Proc Natl Acad Sci USA
,
89
:
10872
-6,  
1992
.