| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
) Can Accelerate ErbB-2-Mediated Mammary Tumorigenesis but Suppresses Tumor Invasion
1 MOBIX, McMaster University, Hamilton, Ontario, Canada; 2 Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada; 3 University of California at Davis, Davis, California; and 4 Molecular Oncology Group, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) 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. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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/PKB. Akt-1, which has two mammalian homologues, Akt-2/PKBß and Akt-3/PKB
, 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.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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). ß-casein transcript was amplified using primers ß-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-3ß, 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).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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. 1A)
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. 1A)
. As expected, the MMTV/AktDD-7 female mice failed to develop mammary tumors during the observation period.
|
, 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.
|
|
than those of NDL2–5 animals at end point (Fig. 3B)
. 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. 2F)
. 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. 3B)
, the margins of the end point late stage bitransgenic lesions are pushing, and the lesions thus do not appear to be invasive (Fig. 3D)
. 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. 1B)
. 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. 4A
, Lanes 2–3 and 8–11) but not in negative control samples from either FVB or NDL2–5 mice (Fig. 4A
, 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. 4A
, 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. 4A
, 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. 4A
, Lane 8) relative to 8–10-week-old NDL2–5 mammary tissue.
|
. 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. 4A)
.
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. 1A)
. 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. 5C)
. 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).
|
|
, 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.
|
. 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.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. 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-3ß (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.
|
ACKNOWLEDGMENTS |
|---|
and 3
. |
FOOTNOTES |
|---|
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.
Received 11/ 5/03. Revised 2/19/04. Accepted 2/20/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B pathway. J Biol Chem, 275: 8027-31, 2000. 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. (HIF-1) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol, 21: 3995-4004, 2001.This article has been cited by other articles:
|
|
 |
|
  K. Kalinsky, L. M. Jacks, A. Heguy, S. Patil, M. Drobnjak, U. K. Bhanot, C. V. Hedvat, T. A. Traina, D. Solit, W. Gerald, et al. PIK3CA Mutation Associates with Improved Outcome in Breast Cancer Clin. Cancer Res., August 15, 2009; 15(16): 5049 - 5059. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maurer, T. Su, L. H. Saal, S. Koujak, B. D. Hopkins, C. R. Barkley, J. Wu, S. Nandula, B. Dutta, Y. Xie, et al. 3-Phosphoinositide-Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma Cancer Res., August 1, 2009; 69(15): 6299 - 6306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Dillon, R. Marcotte, B. T. Hennessy, J. R. Woodgett, G. B. Mills, and W. J. Muller Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression Cancer Res., June 15, 2009; 69(12): 5057 - 5064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Yi, J. W. Chang, W. Han, J. W. Lee, E. Ko, D. H. Kim, J.-Y. Bae, J. Yu, C. Lee, M.-H. Yu, et al. Autoantibody to Tumor Antigen, Alpha 2-HS Glycoprotein: A Novel Biomarker of Breast Cancer Screening and Diagnosis Cancer Epidemiol. Biomarkers Prev., May 1, 2009; 18(5): 1357 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yoeli-Lerner, Y. R. Chin, C. K. Hansen, and A. Toker Akt/Protein Kinase B and Glycogen Synthase Kinase-3{beta} Signaling Pathway Regulates Cell Migration through the NFAT1 Transcription Factor Mol. Cancer Res., March 1, 2009; 7(3): 425 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Stemke-Hale, A. M. Gonzalez-Angulo, A. Lluch, R. M. Neve, W.-L. Kuo, M. Davies, M. Carey, Z. Hu, Y. Guan, A. Sahin, et al. An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer Cancer Res., August 1, 2008; 68(15): 6084 - 6091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Fereshteh, M. T. Tilli, S. E. Kim, J. Xu, B. W. O'Malley, A. Wellstein, P. A. Furth, and A. T. Riegel The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice Cancer Res., May 15, 2008; 68(10): 3697 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Pew, M. Zou, D. R. Brickley, and S. D. Conzen Glucocorticoid (GC)-Mediated Down-Regulation of Urokinase Plasminogen Activator Expression via the Serum and GC Regulated Kinase-1/Forkhead Box O3a Pathway Endocrinology, May 1, 2008; 149(5): 2637 - 2645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Z. Cheng, W. Zhang, and L.-H. Wang Regulation of Cancer Cell Survival, Migration, and Invasion by Twist: AKT2 Comes to Interplay Cancer Res., February 15, 2008; 68(4): 957 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Friedland, J. N. Lakins, M. G. Kazanietz, J. Chernoff, D. Boettiger, and V. M. Weaver {alpha}6beta4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-{kappa}B-dependent resistance to apoptosis in 3D mammary acini J. Cell Sci., October 15, 2007; 120(20): 3700 - 3712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Crowell, V. E. Steele, and J. R. Fay Targeting the AKT protein kinase for cancer chemoprevention Mol. Cancer Ther., August 1, 2007; 6(8): 2139 - 2148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Blanco-Aparicio, O. Renner, J. F.M. Leal, and A. Carnero PTEN, more than the AKT pathway Carcinogenesis, July 1, 2007; 28(7): 1379 - 1386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Ju, S. Katiyar, C. Wang, M. Liu, X. Jiao, S. Li, J. Zhou, J. Turner, M. P. Lisanti, R. G. Russell, et al. Akt1 governs breast cancer progression in vivo PNAS, May 1, 2007; 104(18): 7438 - 7443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Salah, M. Maoz, E. Pokroy, M. Lotem, R. Bar-Shavit, and B. Uziely Protease-Activated Receptor-1 (hPar1), A Survival Factor Eliciting Tumor Progression Mol. Cancer Res., March 1, 2007; 5(3): 229 - 240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shinohara, Y. J. Chung, M. Saji, and M. D. Ringel AKT in Thyroid Tumorigenesis and Progression Endocrinology, March 1, 2007; 148(3): 942 - 947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Blanco-Aparicio, L. Perez-Gallego, B. Pequeno, J. F.M. Leal, O. Renner, and A. Carnero Mice expressing myrAKT1 in the mammary gland develop carcinogen-induced ER-positive mammary tumors that mimic human breast cancer Carcinogenesis, March 1, 2007; 28(3): 584 - 594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. G. Maroulakou, W. Oemler, S. P. Naber, and P. N. Tsichlis Akt1 Ablation Inhibits, whereas Akt2 Ablation Accelerates, the Development of Mammary Adenocarcinomas in Mouse Mammary Tumor Virus (MMTV)-ErbB2/Neu and MMTV-Polyoma Middle T Transgenic Mice Cancer Res., January 1, 2007; 67(1): 167 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Sawyers Will kinase inhibitors have a dark side? N. Engl. J. Med., July 20, 2006; 355(3): 313 - 315. [Full Text] [PDF]
|
|
|
|
|
|
A. Toker and M. Yoeli-Lerner Akt Signaling and Cancer: Surviving but not Moving On. Cancer Res., April 15, 2006; 66(8): 3963 - 3966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Liu, D. C. Radisky, C. M. Nelson, H. Zhang, J. E. Fata, R. A. Roth, and M. J. Bissell Mechanism of Akt1 inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2. PNAS, March 14, 2006; 103(11): 4134 - 4139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Y. Irie, R. V. Pearline, D. Grueneberg, M. Hsia, P. Ravichandran, N. Kothari, S. Natesan, and J. S. Brugge Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition J. Cell Biol., December 19, 2005; 171(6): 1023 - 1034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Fayard, L. A. Tintignac, A. Baudry, and B. A. Hemmings Protein kinase B/Akt at a glance J. Cell Sci., December 15, 2005; 118(24): 5675 - 5678. [Full Text] [PDF]
|
|
|
|
 |
|
 J.-w. Cui, W.-h. Li, J. Wang, A.-l. Li, H.-y. Li, H.-x. Wang, K. He, W. Li, L.-h. Kang, M. Yu, et al. Proteomics-based Identification of Human Acute Leukemia Antigens That Induce Humoral Immune Response Mol. Cell. Proteomics, November 1, 2005; 4(11): 1718 - 1724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wollmann, M. L. Goodman, P. Bhat-Nakshatri, H. Kishimoto, R. J. Goulet Jr, S. Mehrotra, A. Morimiya, S. Badve, and H. Nakshatri The macrophage inhibitory cytokine integrates AKT/PKB and MAP kinase signaling pathways in breast cancer cells Carcinogenesis, May 1, 2005; 26(5): 900 - 907. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
