The Akt/mammalian target of rapamycin (mTOR)/4E-BP1 pathway is considered to be a central regulator of protein synthesis, involving the regulation of cell proliferation, differentiation, and survival. The inhibitors of mTOR as anticancer reagents are undergoing active evaluation in various malignancies including breast cancer. However, the activation status of the Akt/mTOR/4E-BP1 pathway and its potential roles in breast cancers remain unknown. Thus, we examined 165 invasive breast cancers with specific antibodies for the phosphorylation of Akt, mTOR, and 4E-BP1 by immunohistochemistry and compared them with normal breast epithelium, fibroadenoma, intraductal hyperplasia, and ductal carcinoma in situ. We discovered that the phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively from normal breast epithelium to hyperplasia and abnormal hyperplasia to tumor invasion. Phosphorylated Akt, mTOR, and 4E-BP1 were positively associated with ErbB2 overexpression. Survival analysis showed that phosphorylation of each of these three markers was associated with poor disease-free survival independently. In vitro, we further confirmed the causal relationship between ErbB2 overexpression and mTOR activation, which was associated with enhanced invasive ability and sensitivity to a mTOR inhibitor, rapamycin. Our results, for the first time, demonstrate the following: (a) high levels of phosphorylation of Akt, mTOR, and 4E-BP1 in breast cancers, indicating activation of the Akt/mTOR/4E-BP1 pathway in breast cancer development and progression; (b) a link between ErbB2 and the Akt/mTOR/4E-BP1 pathway in breast cancers in vitro and in vivo, indicating the possible role of Akt/mTOR activation in ErbB2-mediated breast cancer progression; and (c) a potential role for this pathway in predicting the prognosis of patients with breast cancer, especially those treated with mTOR inhibitors.

Breast cancer emerges through a multistep process progressing from hyperplasia to premalignant change, in situ carcinoma, and invasive breast cancer, with a gain of oncogene functions and loss of tumor suppressor gene functions (1). It continues to be a major cause of premature death in women, despite progress in early detection, treatment, and advances in our understanding of the molecular basis of breast cancer (2). We urgently need to unveil the exact mechanism of tumor progression and develop new strategies for treatment. Recent research into oncogenic kinase signaling pathways and several specific inhibitors have provided a better understanding of tumorigenesis and targets for developing effective therapeutic strategies (3). The mammalian target of rapamycin (mTOR), also named FRAP (FK506-binding protein 12 and rapamycin-associated protein), RAFT1 (rapamycin and FKBP12 target-1), RAPT1 (rapamycin target-1), or SEP (sirolimus effector protein), is a 289-kDa serine/threonine kinase (4, 5). Because the COOH terminus of mTOR is highly homologous to the catalytic domain of phosphatidylinositol 3′-kinase (PI3K), mTOR is considered to be a member of the PI3K-related protein kinase family (5). Regulated by the upstream molecules PI3K/Akt, mTOR subsequently phosphorylates two downstream substrates, a translation repressor 4E-BP1 (eIF4E-binding protein-1) and ribosomal p70S6K, resulting in inactivation of the former and activation of the latter, thus initiating protein translation (6, 7). PI3K/Akt/mTOR has been considered a central regulatory pathway of protein translation involved in the regulation of cell proliferation, growth, differentiation, migration, and survival (8, 9, 10, 11). Activation of Akt and its prognostic value in breast cancers have been reported (12, 13, 14); however, whether mTOR and 4E-BP are constitutively activated in breast cancers has not been reported. Because ErbB2 overexpression, which has been found in approximately 30% of human breast cancers (15), can activate Akt/PKB (13, 16, 17, 18, 19), we hypothesized that mTOR and its downstream element 4E-BP1 might be activated by ErbB2 overexpression. We investigated the relationship between ErbB2 overexpression and the phosphorylation of mTOR and 4E-BP1 in vitro and in vivo.

Rapamycin, a specific inhibitor of mTOR, and its analogs (such as CCI-779, RAD001, and AP23573) have demonstrated prominent growth-inhibitory effects against a broad range of human cancers in both preclinical and early clinical investigations (7, 10, 20). However, there exists an urgent need to determine whether tumors with specific molecular abnormalities may be hypersensitive or hyper-resistant to rapamycin (7) and whether the inhibitory ability of rapamycin differs in breast cancers with different mTOR/4E-BP1 phosphorylation levels. Answering these questions might facilitate the future development of diagnostic and therapeutic strategies.

This study focused on the activation of three important molecules in the Akt/mTOR/4E-BP1 pathway, Akt, mTOR, and 4E-BP1. We first detected the phosphorylation status of Akt, mTOR, and 4E-BP1 in 165 invasive breast cancers, compared with normal breast epithelium, fibroadenoma, intraductal hyperplasia (IDH), and ductal carcinoma in situ (DCIS), using immunohistochemistry with specific antibodies. We then analyzed the relationship of Akt, mTOR, and 4E-BP1 with ErbB2 overexpression and other clinical characteristics. Additionally, we investigated the association between phospho (p)-mTOR and p-4E-BP1 expression levels and invasive ability with or without rapamycin treatment in two ErbB2-transfected breast cancer cell lines and their parental counterparts, MDA-MD-435 (21) and MCF7. We discovered that phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively as normal breast epithelium developed into epithelial hyperplasia, abnormal hyperplasia, and tumor invasion, and each phosphorylation level positively associated with one another, indicating that the activation of the Akt/mTOR/4E-BP1 pathway is involved in breast cancer development and progression. Higher phosphorylation levels of the three markers were associated with ErbB2 overexpression and predicted poor survival. In vitro, we further confirmed the association between ErbB2 overexpression and mTOR activation, which enhanced invasive ability as well as sensitivity to rapamycin.

Patient Population and Tissue Samples.

A total of 165 primary invasive breast cancer specimens obtained from the Department of Pathology, Shanghai Cancer Hospital, Fudan University were studied retrospectively. The patients were diagnosed between 1988 and 1991 and had modified radical mastectomy and were treated with postoperative adjuvant therapy. Clinical data were obtained from medical records. Patients were followed-up for a median of 76.4 months after their initial cancer surgery. A total of 58 patients experienced recurrences, and there have been 41 deaths. In addition, 8 normal breast tissues, 8 fibroadenomas, 14 IDHs (including 4 atypical ductal hyperplasias, which were combined with IDH because the case numbers were limited), and 12 cases of DCIS were included in the study for comparison. Archival formalin-fixed, paraffin-embedded tumor specimens were stained with hematoxylin and eosin to confirm the diagnosis. A representative block was chosen for the study for each case.

Antibodies.

The DAKO Hercep Test for ErbB2 immunohistochemistry and Envision+ systems were purchased from DAKO (Carpinteria, CA). Monoclonal antibody c-ErbB2/c-Neu (Ab-3) was purchased from Oncogene (San Diego, CA) for Western blotting. Polyclonal antibodies against p-Akt (Ser473), p-mTOR (Ser2448), p-4E-BP1 (Ser65), and p-p70 S6 kinase [p70S6K (Thr389; 1A5)] were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Horseradish peroxidase-labeled antimouse and antirabbit antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

Immunohistochemistry and Evaluation.

Immunohistochemical staining was performed using an immunoperoxidase technique as described previously (22). Briefly, after deparaffinization and rehydration, 4-μm sections were subjected to heat-induced epitope retrieval in 0.01 mol/L citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked for 5 minutes in 3% hydrogen peroxide. Nonspecific binding was blocked by treatment with a blocking reagent (DAKO) for 20 minutes at room temperature. The slides were incubated with primary antibody for 2 hours at room temperature or overnight at 4°C. Primary antibodies included ErbB2, p-Akt (1:50), p-mTOR (1:50), p-4E-BP1 (1:100), and p-p70S6K (1:800). Immunodetection was performed with the DAKO Hercep Test system for ErbB2 and with the Envision+ system for p-mTOR (Ser2448), p-4E-BP1 (Ser65), p-Akt (1:50), and p-p70S6K (1:800). Next, 3–3′-diaminobenzidine was used for color development, and hematoxylin was used for counterstaining. Slides processed with normal rabbit serum (DAKO) in place of the primary antibody were used as negative controls, and slides of tissues known to express p-Akt, p-mTOR, p-4E-BP1, and p-p70S6K were used as positive controls in each staining.

Strong and complete membrane staining in >10% of the tumor cells was defined as ErbB2 overexpression or ErbB2 high expression (22). The p-Akt, p-mTOR, p-4E-BP1, and p-p70S6K levels were grouped into three categories based on both staining intensity and positive frequency according to a previously described scoring method with a slight modification (12). Tumors with <10% cells with weak staining were scored as 0, tumors with >10% of cells with weak staining or <20% of cells with strong staining were scored as 1, and tumors with >20% of cells with strong staining were scored as 2.

Cell Lines and Cell Cultures.

The MDA-MB-435, MCF7, and BT474 human breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Wild-type ErbB2 transfectants of the two cell lines MDA-MB-435.eB and MCF7.eB were established as described previously (23, 24). The cells were maintained in Dulbecco’s modified Eagle’s medium containing high glucose (DMEM/F-12; Life Technologies, Inc., Grand, NY) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 and 95% air at 37°C. Cells were passaged by treatment with a solution containing 0.25% trypsin and 1 mmol/L EDTA when cells reached 80% confluence.

Rapamycin Treatment and Western Blot Analysis.

Subconfluent MCF7 and MCF7.eB cells were incubated with 30 nmol/L rapamycin (Cell Signaling Technology, Inc.) for 16 hours, and subconfluent MDA-MB-435 and MDA-MB-435.eB cells were incubated with 60 nmol/L rapamycin for 16 hours. Untreated cells were used as controls. Cell lysates were collected, and Western blot analysis was performed as described previously (25). The membranes were probed with specific antibodies against ErbB2, p-mTOR, p-4E-BP1, or β-actin.

ErbB2 RNA Interference.

To target specific regions of ErbB2, two oligonucleotides were synthesized: 5′-AAGTACACGATGCGGAGACTGCCTGTCTC-3′ and 5′-AACAGTCTCCGCATCGTGTACCCTGTCTC-3′. These oligonucleotides were the starting material for the Silencer small interfering RNA (siRNA) construction kit (Ambion), and the in vitro transcription reaction was carried out, resulting in double-stranded siRNA. For RNA interference, a subline of the BT474 breast cancer cell line was cultured to ∼30% confluence and then transfected with various concentrations of ErbB2 siRNA. Three hours after transfection, cells were rescued with serum-containing medium, and cell lysates were collected for Western blot analysis 48 hours after transfection. ErbB2 siRNA (66.7 nmol/L) was able to significantly decrease the ErbB2 protein level compared with green fluorescent protein siRNA- or mock-transfected cells.

In vitro Cell Invasion Assay.

The invasion assay was performed as described previously (26). Polycarbonate membrane invasion chambers from 24-well Transwell plates (Corning Inc., Corning NY) were coated with a 1:80 dilution (for MCF7 and MCF7.eB cells) or a 1:40 dilution (for MDA-MB-435 and MDA-MB435.eB cells) Matrigel matrix (BD Biosciences, Bedford, MA) and allowed to dry overnight. Cells with or without rapamycin treatment (30 nmol/L for MCF7 and MCF7.eB cells; 60 nmol/L for MDA-MB-435 and MDA-MB-435.eB cells) were harvested after 3 hours, and 2 × 106 cells in 100 μL of DMEM/F-12 plus 10% FBS with the same concentration of rapamycin were loaded into the upper compartment and incubated overnight at 37°C. The lower compartment of the Transwell contained 0.6 mL of DMEM/F-12 plus 10% FBS. The cells were fixed with 3% glutaraldehyde and stained with Giemsa. The cells on the upper surface of the filter were removed, and the invasive abilities of the cells were determined by counting the number of cells per high-power field (×200) that had migrated through the filter. Each sample was assayed in triplicate, and the assay was repeated at least twice.

Statistical Analysis.

Cochran-Armitage trend test was used to test for trends in binomial proportions across levels of a single factor. The χ2 test was used to investigate the independence of categorical variables. The nonparametric Wilcoxon test was used to assess the independence between two continuous variables without assuming any distribution assumption. Disease-free survival (DFS) was dated from the date of surgery to relapse date or last follow-up date. Patients who died from breast cancer with no recurrence date specified were counted as disease events at their date of death. DFS was estimated using the Kaplan-Meier product-limit method. The two-sided log-rank test was used to test the association between variables and survivals. All P values presented are two-sided. The cutoff for significance was set at P ≤ 0.05. Statistical analyses were carried out using SAS 8.0 and Splus 6.0.

Increased Phosphorylation of Akt/mTOR/4E-BP1 during Tumor Development and Progression.

To investigate the potential role of the Akt/mTOR/4E-BP1 pathway activation in breast cancer development and progression, we examined phosphorylation of Akt, mTOR, and 4E-BP1 in a series of breast tissue samples representing different stages (8 normal breast tissues, 8 fibroadenomas, 14 IDHs, 12 cases of DCIS, and 165 invasive breast cancers). Akt is activated by both translocation to the plasma membrane and phosphorylation at Thr308 and Ser473(27, 28). Akt has a wide range of substrates, such as ASk1, Bad, mTOR, the Forkhead family, and IκB kinase (27, 28). Mammalian target of rapamycin was found to be phosphorylated at Ser2448 directly by Akt or through growth factor stimulation, such as by insulin, by increased amino acid levels, or by exercise recovery via Akt (29, 30, 31). Phosphorylation of mTOR at Ser2448 has been suggested to be an important marker for the activation of the pathway (29, 30, 32). Additionally, mTOR can phosphorylate one of its downstream substrates, 4E-BP1, on the inhibitory phosphorylation site, Ser65. Thus, p-4E-BP1 (Ser65) is considered an indication of mTOR activation (33). Therefore, we performed immunohistochemical studies using three antibodies specific for p-Akt (Ser473), p-mTOR (Ser2448), and p-4E-BP1 (Ser65). We found that the phosphorylation levels of all three molecules generally increased as disease progressed from epithelial proliferation to abnormal proliferation to invasion (Table 1; Fig. 1). None of the eight fibroadenomas showed expression of any of these three markers. Of the eight normal breast tissues, only two samples had low p-Akt, one sample had high p-mTOR, and none of samples had p-4E-BP1; however, in IDH and DCIS samples, p-Akt, p-mTOR, and p-4E-BP1 expression was dramatically increased. High p-Akt, p-mTOR, and p-4E-BP1 expression was seen in 5 of 14 (35.7%), 4 of 14 (28.6%), and 2 of 14 (14.3%) samples of IDH, respectively, and 5 of 12 (41.7%), 4 of 12 (33.3%), and 2 of 12 (16.7%) samples of DCIS, respectively. In invasive breast carcinomas, there were different expression levels in different patients (Fig. 2); 69 of 165 samples (41.8%), 70 of 165 samples (42.4%), and 68 of 165 samples (41.2%) had high levels of p-Akt, p-mTOR, and p-4E-BP1, respectively. The Cochran-Armitage trend test was used to determine whether there was a trend in proportions of high p-Akt, p-mTOR, or p-4E-BP1 across the five groups. The results supported the trend that percentages of high phosphorylation of Akt, mTOR, and 4E-BP1 increased as the proliferation and invasion increased (Table 1; P < 0.01). In addition, we found that the phosphorylation of each pair of molecules in invasive breast cancers was highly positively correlated using χ2 analysis (P < 0.01; data not shown), which is consistent with in vitro studies on the causal association of the molecules (6, 7).

Phosphorylation of Akt/mTOR/4E-BP1 Is Associated with ErbB2 Overexpression in Invasive Breast Cancers.

Because the overexpression of ErbB2 has been implicated in breast cancer progression and therapeutic resistance and because Akt can be activated by ErbB2 overexpression in transformed cells (16, 25, 34), we investigated whether Akt/mTOR/4E-BP1 phosphorylation was associated with ErbB2 overexpression. Notably, the proportion of ErbB2-overexpressing breast cancers increased as the phosphorylation of Akt, mTOR, and 4E-BP1 increased, which suggests that high phosphorylation of Akt, mTOR, and 4E-BP1 is more likely to occur in ErbB2-overexpressing breast tumors (P = 0.03, P < 0.01, and P < 0.01, respectively; Fig. 2; Table 2). We next examined whether Akt/mTOR/4E-BP1 are also associated with other clinicopathological variables, including estrogen receptor (ER) status, stage, grade, lymph node metastasis, patient age, and tumor size. We found that p-Akt was positively correlated with ER expression (P < 0.02), and this result was consistent with data from previous in vitro studies (17, 18, 35). However, there is no strong evidence of any association between phosphorylation of the three molecules and other clinicopathological variables in breast carcinoma (Table 2). In addition, we found that patients who had ErbB2 overexpression tended to have larger tumors, negative ER status, and a higher tumor grade (P = 0.02, P < 0.01, and P = 0.02; data not shown), which are consistent with previous reports.

Prognostic Value of Phosphorylation of Akt/mTOR/4E-BP1 in Invasive Breast Cancers.

Because ErbB2 overexpression predicts poor patient survival (22, 36) and because we found that ErbB2 overexpression was positively correlated with p-Akt, p-mTOR, and p-4E-BP1 in our data set, we investigated whether p-Akt, p-mTOR, and p-4E-BP1 might be associated with poor prognosis in the 165 patients with invasive breast carcinoma. We performed univariate survival analysis to show the associations of DFS with p-Akt, p-mTOR, and p-4E-BP1 and with other clinical characteristics (Table 3). In addition to ErbB2, tumor grade, and nodal status, which are established prognostic factors (37, 38), p-Akt, p-mTOR, and p-4E-BP1 were also statistically associated with DFS. Patients whose tumors had higher mTOR phosphorylation had significantly shorter DFS (P < 0.01). Patients whose tumors had lower Akt or 4E-BP1 phosphorylation tended to have longer DFS. Kaplan-Meier survival curves demonstrated the association between DFS and these markers (Fig. 3). Additionally, there was also a tendency for patients with large, ER-negative, high-stage tumors to have poor DFS, although the difference was not statistically significant in this patient group (Table 3).

Increased mTOR and 4E-BP1 Phosphorylation and Enhanced Sensitivity to Rapamycin in ErbB2-Overexpressing Cells Compared with Parental Cells.

Because we observed that the expression of p-Akt and its downstream molecules p-mTOR and p-4E-BP1 was statistically significantly associated with ErbB2 overexpression in breast cancers and because Akt activation by ErbB2 overexpression has been reported (13, 16, 17, 18, 19), we next directly investigated the relationship between ErbB2 and p-mTOR or p-4E-BP1 in cultured breast cancer cells. To determine whether ErbB2 overexpression would lead to increased p-mTOR and p-4E-BP1, we compared the levels of p-mTOR and p-4E-BP1 by Western blot in the breast cancer cell lines MDA-MB-435 and MCF7, which have low ErbB2 expression, with their ErbB2 transfectants, 435.eB and MCF7.eB, which have high ErbB2 expression (Fig. 4,A). We found that both the ErbB2-overexpressing cell lines 435.eB and MCF7.eB had higher p-mTOR and p-4E-BP1 levels than their parental cells (Fig. 4 A).

Rapamycin, a mTOR-specific inhibitor, functions by first binding to FKBP12 to form a FKBP12-rapamycin complex, and this complex then interacts with the FKBP12-rapamycin-binding domain in mTOR and inhibits the function of mTOR (29). Here we examined whether ErbB2-mediated increases of p-mTOR and p-4E-BP1 would be sensitive to inhibition by rapamycin. Although p-4E-BP1 was not reduced in parental breast cancer cells with low ErbB2 expression, p-4E-BP1 was significantly decreased after rapamycin treatment in cells expressing high levels of ErbB2, indicating high sensitivity to rapamycin in the ErbB2-overexpressing cells (Fig. 4,A). There was no significant change in p-mTOR after rapamycin treatment, which is consistent with the function of rapamycin (Fig. 4,A). Moreover, the BT474 breast cancer cell line, which expressed high levels of ErbB2 protein and has high levels of p-mTOR and p-4E-BP1, had reduced p-mTOR and p-4E-BP1 after ErbB2 expression was down-regulated by ErbB2 RNA interference treatment (Fig. 4,B). This further supports the causal relationship between ErbB2 overexpression and increased mTOR/4E-BP1 phosphorylation. Because we observed the relationship between Akt/mTOR/4E-BP1 phosphorylation and disease recurrence, which was most likely due to cancer metastasis, we investigated the invasion ability and sensitivity to rapamycin in the ErbB2-overexpressing MCF7.eB transfectants and their parental cells by in vitro invasion assay (Fig. 4,C). Compared with the parental MCF7 cells, ErbB2-overexpressing MCF7.eB cells had higher invasion ability; however, after rapamycin treatment, both cell lines had reduced invasion. The reduction in the number of invasive cells in MCF7.eB cells (about 60% reduction) is greater than that in MCF7 cells (about 40% reduction; Wilcoxon test, P < 0.05; Fig. 4 C), indicating that the ErbB2-overexpressing MCF7.eB cells have higher sensitivity to rapamycin than the MCF7 cells (P < 0.05).

Because the clinical efficacy of the mTOR inhibitor rapamycin is measured by the phosphorylated levels of its downstream substrate, p70S6K(39), we also examined whether the phosphorylation of p70S6K is associated with p-Akt, p-mTOR, and p-4E-BP1. The results show that p-p70S6K levels are positively associated with p-mTOR, p-AKT, and p-4E-BP1 (Table 4). This indicates that tumors with negative p-p70S6K are more likely to have negative p-mTOR, p-AKT, and p-4E-BP1 and that tumors with overexpression of p-p70S6K are more likely to overexpress p-mTOR, p-AKT, and p-4E-BP1.

Using immunohistochemistry with three phosphorylation-specific antibodies to detect Akt, mTOR, and 4E-BP1 phosphorylation status in breast cancers, we found that increased Akt/mTOR/4E-BP1 phosphorylation occurred at the stage of IDH that includes atypical ductal hyperplasia, which has a high possibility of developing into breast cancer (1), and the phosphorylation levels were even higher in DCIS and invasive breast cancers (Table 1; Cochran-Armitage trend test, P < 0.01). We also found that each pair of phosphorylated proteins were highly positively associated in invasive breast cancers, i.e., tumors with higher p-Akt were more likely to have higher p-mTOR or p-4E-BP1 expression; tumors with higher p-mTOR were more likely to have higher p-4E-BP1 expression (χ2 test, P < 0.01; data not shown), confirming the activation cascade of the Akt/mTOR/4E-BP1 pathway in breast cancers. As reported in mammalian cells, translation rates are often correlated with eIF4E (eukaryotic initiation factor 4E) activity (4), which directs the translation machinery via an interaction with one of two large scaffolding proteins, termed eIF4GI and eIF4GII. This interaction is regulated by the eIF4E-binding proteins (4E-BPs). The binding of 4E-BPs to eIF4E is regulated by phosphorylation; that is, hyperphosphorylation of the 4E-BPs abrogates this interaction, resulting in activation of eIF4E, whereas hypophosphorylated 4E-BPs bind avidly to eIF4E, resulting in its inactivation (4, 40). The reduction of protein synthesis was associated with decreased activation of protein kinases in the mTOR signal pathway, as shown by reduced phosphorylation of Akt, mTOR, and 4E-BP1 (41). In our study, none of the eight fibroadenomas and only a small part of normal breast epithelium had p-Akt–, p-mTOR–, or p-4E-BP1–positive staining, indicating low activity of the Akt/mTOR/4E-BP1 pathway in these quiescent or low-proliferating cells. We found high levels of p-Akt, p-mTOR, and p-4E-BP1 in the IDH and DCIS abnormal proliferative cells, and because breast cancer is the result of a multistep process from hyperplasia to atypical hyperplasia to DCIS and to invasive breast cancer (1), we believe that activation of the Akt/mTOR/4E-BP1 pathway might be an early event in breast epithelium oncogenic transformation and thus involved in breast cancer development and progression. Because a large number of tumors, including breast cancers, have increased levels of eIF4E (4), activation of Akt/mTOR/4E-BP1 might be involved in the abnormal initiation of protein synthesis. The underlying mechanism of how the Akt/mTOR/4E-BP1 pathway is activated and how it affects breast epithelium transformation and progression toward cancer development needs to be further investigated.

Studies of patients with ErbB2-overexpressing tumors have shown that they have a significantly worse clinical outcome than patients whose tumors do not overexpress ErbB2 (15, 22). However, how ErbB2 exerts its function through its downstream signaling molecules still needs intensive investigation. A connection has been made between ErbB2 overexpression and up-regulation of Akt expression (13, 16, 17, 18, 19). Whether mTOR or 4E-BP1 phosphorylation is also associated with ErbB2 overexpression in patients with breast cancer is unclear. In our study, elevated expression of ErbB2 was indeed correlated with increased phosphorylation of Akt, mTOR, and 4E-BP1 in patients with invasive breast cancers. We further investigated this relationship in two different cell lines, MDA-MB-435 and MCF7, which have different endogenous genetic backgrounds, and in their ErbB2-transfected counterparts. We found that ErbB2 overexpression had increased p-mTOR and p-4E-BP1 in both cell lines. The results suggested a general trend that frequent activation of the Akt/mTOR/4E-BP1 pathway in breast cancers is at least partly due to ErbB2 overexpression. Because the percentages of Akt, mTOR, and 4E-BP1 phosphorylation were higher than that of ErbB2 overexpression in breast cancers, there must be other factors regulating Akt/mTOR/4E-BP1 pathway activation. A better understanding of the mechanisms regulating the pathway will open the possibility of selective control of the pathway and identification of a new generation of more effective drugs for breast cancer treatment.

We also found that higher levels of p-AKT, p-mTOR, and p-4E-BP1 were statistically significantly associated with poor DFS. Patients whose tumors had higher p-Akt, p-mTOR, or p-4E-BP1 levels tended to have shorter DFS; on the other hand, patients whose tumors had lower Akt, mTOR, or 4E-BP1 phosphorylation were more likely to be free of recurrence at the 5-year follow up. It would also be interesting to investigate whether the activation of mTOR/4E-BP1 correlates with resistance to chemotherapeutic treatments given to patients with invasive breast cancer. The majority of patients in this sample set were treated with cyclophosphamide, methotrexate, and 5-fluoroucil (CMF) chemotherapy after undergoing surgery. Detailed response data to chemotherapeutics (such as complete response, partial response, or no response) are unavailable; however the DFS results may indirectly reflect that the activation of mTOR/4E-BP1 was correlated with resistance to CMF chemotherapy. Thus, the detection of p-Akt, p-mTOR, or p-4E-BP1 could be useful for predicting tumor progression, particularly when the classical survival parameters are insufficient, such as in patients with small, nonmetastatic breast cancer. Because metastasis is the major factor leading to breast cancer progression and eventually to patient death, we compared the invasion ability and response to rapamycin in ErbB2-overexpressing cell lines, which we found to have higher mTOR and 4E-BP1 phosphorylation than their parental cell lines. These ErbB2-overexpressing cell lines with higher mTOR and 4E-BP1 phosphorylation also had high invasion ability, which could be effectively inhibited by the mTOR inhibitor rapamycin (Fig. 4,C). It will be interesting to investigate in the future whether rapamycin can inhibit breast cancer metastasis in patients. Malignancies driven by the stimulation of receptors that constitutively activate PI3K/Akt/mTOR-related pathways may be particularly dependent on this pathway for growth and therefore may be especially sensitive to rapamycin analogs. For example, PTEN-deficient cells are remarkably more sensitive to mTOR inhibition than PTEN wild-type cells (42, 43). This might be due to elevated phosphorylated Akt or phosphatidylinositol 3,4,5-trisphosphate levels in cells with loss of PTEN, raising the possibility that the sustained activation of the signaling molecules renders cells more dependent on this pathway for growth and that the cells could be more sensitive to mTOR inhibitors. We found that ErbB2-overexpressing cells had enhanced sensitivity to rapamycin treatment (Fig. 4 C). Similarly, ErbB2-overexpressing cells with the activated Akt/mTOR/4E-BP1 pathway may be more dependent on this pathway for growth and therefore more sensitive to mTOR inhibition, thus expanding on previous research that has shown rapamycin sensitivity to cells with active Akt (44) and correlations with ErbB2 overexpression (20). In addition to our findings of the correlation of the activation of the Akt/mTOR/4E-BP1 pathway in primary tumor samples, our investigation also showed a correlation with the phosphorylation status of the other main target of mTOR, p70S6K. By showing this correlation, we show that both targets of mTOR are involved in the pathway examined, thereby increasing the power and effectiveness of a mTOR inhibitor by eliminating both sides of the downstream pathway. The effect of a mTOR inhibitor, CCI-779, has been investigated on a panel of breast cancer cell lines. Six of eight cell lines studied were sensitive, and two cell lines were resistant. Sensitive lines were estrogen dependent (MCF7, BT474, and T47D), lacked expression of the tumor suppressor PTEN (MDA-MB-468 and BT-549), and/or overexpressed the ErbB2 oncogene (SKBR-3 and BT-474). Resistant cell lines (MDA-MB-435 and MDA-MB-231) shared none of these properties (7). Interestingly, although they were derived from MDA-MB-435 cells, our ErbB2-overexpressing 435.eB cells were sensitive to rapamycin. This further supports the notion that ErbB2-overexpressing cells are sensitive to mTOR inhibitors. Taken together, ErbB2 overexpression as well as PTEN loss may be involved in regulation of the Akt/mTOR/4E-BP1 pathway and eventually affect sensitivity to mTOR inhibitors. Mammalian target of rapamycin inhibitors have been used as anticancer drugs in phase I and II clinical trials, demonstrating promising anticancer activity but relatively mild side effects. Prospectively identifying patients who are unlikely to respond to the inhibitor of rapamycin can spare them the potential side effects and unnecessary cost. Although measurement of the phosphorylation status of 4E-BP1 or p70S6K has been raised as a possible factor to determine drug dose or evaluate its effectiveness (11), there are no verified factors that can be used to predict response to mTOR inhibitors. These in vitro findings are preliminary in nature and require further validation in vivo; however, from our study, frequent deregulation of the Akt/mTOR/4E-BP1 signaling pathway and its prognostic role in breast cancers support the notion of using mTOR inhibitors as an additional breast cancer treatment. Moreover, the detection of p-Akt, p-mTOR, or p-4E-BP1 through the simplicity and reproducibility of immunohistochemical stainings might be very helpful for identifying and predicting which patients are most likely to derive the most benefit from treatment with a mTOR inhibitor.

Fig. 1.

Representative immunohistochemistry staining of p-Akt, p-mTOR, and p-4E-BP1 in normal breast epithelium (A–C) fibroadenoma (D–F), IDH (G–I), and DCIS (J–L). Tissues were stained with specific antibodies for p-Akt (Ser473), p-mTOR (Ser2448), and p-4E-BP1 (Ser65). Original magnification, ×100.

Fig. 1.

Representative immunohistochemistry staining of p-Akt, p-mTOR, and p-4E-BP1 in normal breast epithelium (A–C) fibroadenoma (D–F), IDH (G–I), and DCIS (J–L). Tissues were stained with specific antibodies for p-Akt (Ser473), p-mTOR (Ser2448), and p-4E-BP1 (Ser65). Original magnification, ×100.

Close modal
Fig. 2.

Representative immunohistochemistry stainings of ErbB2, p-Akt, p-mTOR, and p-4E-BP1 in invasive breast cancers. The staining of p-Akt, p-mTOR, and p-4E-BP1 was higher in ErbB2-overexpressing breast carcinomas. A–D show one case with negative ErbB2, p-Akt, p-mTOR, and p-4E-BP1. E–H show one case with low ErbB2, p-Akt, p-mTOR, and p-4E-BP1. I–L show one case with high ErbB2, p-Akt, p-mTOR, and p-4E-BP1. Original magnification, ×100.

Fig. 2.

Representative immunohistochemistry stainings of ErbB2, p-Akt, p-mTOR, and p-4E-BP1 in invasive breast cancers. The staining of p-Akt, p-mTOR, and p-4E-BP1 was higher in ErbB2-overexpressing breast carcinomas. A–D show one case with negative ErbB2, p-Akt, p-mTOR, and p-4E-BP1. E–H show one case with low ErbB2, p-Akt, p-mTOR, and p-4E-BP1. I–L show one case with high ErbB2, p-Akt, p-mTOR, and p-4E-BP1. Original magnification, ×100.

Close modal
Fig. 3.

Kaplan-Meier DFS curves of breast cancer patients with different p-Akt (A), p-mTOR (B), and p-4E-BP1 (C) status. Patients whose tumors have high levels of p-Akt, p-mTOR, or p-4E-BP1 tend to have worse survival.

Fig. 3.

Kaplan-Meier DFS curves of breast cancer patients with different p-Akt (A), p-mTOR (B), and p-4E-BP1 (C) status. Patients whose tumors have high levels of p-Akt, p-mTOR, or p-4E-BP1 tend to have worse survival.

Close modal
Fig. 4.

Association between ErbB2 overexpression and p-mTOR and p-4E-BP1 and their response to rapamycin. A. As measured by Western blotting, ErbB2-transfected 435.eB and MCF7.eB cells have higher p-mTOR and p-4E-BP1. After rapamycin treatment, p-4E-BP1 decreased, but p-mTOR had no obvious change. B. BT474 cells had high ErbB2 expression. After ErbB2 siRNA treatment, both p-mTOR and p-4E-BP1 decreased, as confirmed by Western blotting. C. ErbB2 overexpression increased invasiveness and sensitivity to rapamycin. MCF7.eB cells had higher invasive ability than their parental cells, but that ability was greatly reduced after rapamycin treatment (Wilcoxon test, P < 0.01). Mean number of invasive cells and SEs are shown.

Fig. 4.

Association between ErbB2 overexpression and p-mTOR and p-4E-BP1 and their response to rapamycin. A. As measured by Western blotting, ErbB2-transfected 435.eB and MCF7.eB cells have higher p-mTOR and p-4E-BP1. After rapamycin treatment, p-4E-BP1 decreased, but p-mTOR had no obvious change. B. BT474 cells had high ErbB2 expression. After ErbB2 siRNA treatment, both p-mTOR and p-4E-BP1 decreased, as confirmed by Western blotting. C. ErbB2 overexpression increased invasiveness and sensitivity to rapamycin. MCF7.eB cells had higher invasive ability than their parental cells, but that ability was greatly reduced after rapamycin treatment (Wilcoxon test, P < 0.01). Mean number of invasive cells and SEs are shown.

Close modal

Grant support: M. D. Anderson Cancer Center Core Grant P30-CA16672 from the National Cancer Institute; National Institutes of Health grants 2R01-CA60488 (D. Yu), DAMD17-01-1-0361 (D. Yu), and DAMD17-02-1-046201 (D. Yu); 5PO1-CA099031 (V. Hawthorne); and the M. D. Anderson BCRP Fund (D. Yu).

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: Dihua Yu, Department of Surgical Oncology, Unit 107, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3636; Fax: 713-794-4830; E-mail: dyu@mdanderson.org

Table 1

Phosphorylation of Akt, mTOR, and 4E-BP1 in different breast tissues

Groupsnp-Akt n (%)p-mTOR n (%)p-4E-BP1 n (%)
012012012
Normal epithelium 6 (75) 2 (25) 0 (0) 7 (87.5) 0 (0) 1 (12.5) 8 (100) 0 (0) 0 (0) 
Fibroadenoma 8 (100) 0 (0) 0 (0) 8 (100) 0 (0) 0 (0) 8 (100) 0 (0) 0 (0) 
IDH 14 3 (21.4) 6 (42.9) 5 (35.7) 7 (50) 3 (21.4) 4 (28.6) 10 (71.4) 2 (14.3) 2 (14.3) 
DCIS 12 1 (8.3) 6 (50) 5 (41.7) 1 (8.3) 7 (58.3) 4 (33.3) 5 (41.7) 5 (41.7) 2 (16.7) 
Invasive breast cancers 165 43 (26.1) 53 (32.1) 69 (41.8) 50 (30.3) 47 (28.5) 68 (41.2) 47 (28.5) 48 (29.1) 70 (42.4) 
P                  *    <0.01   <0.01   <0.01 
Groupsnp-Akt n (%)p-mTOR n (%)p-4E-BP1 n (%)
012012012
Normal epithelium 6 (75) 2 (25) 0 (0) 7 (87.5) 0 (0) 1 (12.5) 8 (100) 0 (0) 0 (0) 
Fibroadenoma 8 (100) 0 (0) 0 (0) 8 (100) 0 (0) 0 (0) 8 (100) 0 (0) 0 (0) 
IDH 14 3 (21.4) 6 (42.9) 5 (35.7) 7 (50) 3 (21.4) 4 (28.6) 10 (71.4) 2 (14.3) 2 (14.3) 
DCIS 12 1 (8.3) 6 (50) 5 (41.7) 1 (8.3) 7 (58.3) 4 (33.3) 5 (41.7) 5 (41.7) 2 (16.7) 
Invasive breast cancers 165 43 (26.1) 53 (32.1) 69 (41.8) 50 (30.3) 47 (28.5) 68 (41.2) 47 (28.5) 48 (29.1) 70 (42.4) 
P                  *    <0.01   <0.01   <0.01 

NOTE. Phorphorylation levels of Akt, mTOR, and 4E-BP1 in different stages of disease progression. Results indicate that phosphorylation levels increase with disease progression.

*

Cochran-Armitage trend test combining 0 and 1 into one group.

Table 2

Association of p-Akt, p-mTOR, and p-4E-BP1 with ErbB2 and patient characteristics

Factornp-AKT n (%)p-mTOR n (%)p-4-EBP1 n (%)
012P012P012P
ErbB2     0.03*    <0.01    <0.01 
 low 112 34 (30) 37 (33) 41 (37)  43 (38) 32 (29) 37 (33)  42 (37) 32 (29) 38 (34)  
 high 53 9 (17) 16 (30) 28 (53)  7 (13) 15 (28) 31 (58)  5 (10) 16 (30) 32 (60)  
ER     0.02*    0.13    0.11 
 Negative 83 26 (31) 30 (36) 27 (33)  27 (32) 28 (34) 28 (34)  29 (35) 22 (26) 32 (39)  
 Positive 82 17 (21) 23 (28) 42 (51)  23 (28) 19 (23) 40 (49)  18 (22) 26 (32) 38 (46)  
Stage     0.45    0.61    0.77 
 1 21 8 (38) 8 (38) 5 (24)  8 (38) 4 (19) 9 (43)  8 (38) 4 (19) 9 (43)  
 2 103 24 (23) 33 (32) 46 (45)  32 (31) 32 (31) 39 (38)  29 (28) 31 (30) 43 (42)  
 3 41 11 (27) 12 (29) 18 (44)  10 (24) 11 (27) 20 (49)  10 (24) 13 (32) 18 (44)  
Grade     0.18    0.34    0.62 
 1 36 12 (33) 10 (28) 14 (39)  14 (39) 7 (19) 15 (42)  13 (36) 9 (25) 14 (39)  
 2 81 19 (24) 22 (27) 40 (49)  19 (24) 26 (32) 36 (44)  19 (23) 24 (30) 38 (47)  
 3 48 12 (25) 21 (44) 15 (31)  17 (35) 14 (29) 17 (35)  15 (31) 15 (31) 18 (38)  
Node     0.99*    0.46    0.69 
 0 57 14 (25) 20 (35) 23 (40)  15 (26) 17 (30) 25 (44)  17 (30) 13 (23) 27 (47)  
 >0 108 29 (27) 33 (30) 46 (43)  35 (32) 30 (28) 43 (40)  30 (28) 35 (32) 43 (40)  
Age (y)  54 (33–74) 50 (30–75) 48 (25–69) 0.32 51 (34–72) 51 (25–69) 48 (29–75) 0.89 49 (30–74) 49 (34–69) 52 (25–75) 0.99 
Tumor size (cm)  3 (0–11) 4 (1.9–8) 3 (1.9–8) 0.41 3 (1.9–8) 4 (1–8) 4 (1–8) 0.29 4 (1.9–8) 4 (1–8) 3 (1–8) 0.67 
Factornp-AKT n (%)p-mTOR n (%)p-4-EBP1 n (%)
012P012P012P
ErbB2     0.03*    <0.01    <0.01 
 low 112 34 (30) 37 (33) 41 (37)  43 (38) 32 (29) 37 (33)  42 (37) 32 (29) 38 (34)  
 high 53 9 (17) 16 (30) 28 (53)  7 (13) 15 (28) 31 (58)  5 (10) 16 (30) 32 (60)  
ER     0.02*    0.13    0.11 
 Negative 83 26 (31) 30 (36) 27 (33)  27 (32) 28 (34) 28 (34)  29 (35) 22 (26) 32 (39)  
 Positive 82 17 (21) 23 (28) 42 (51)  23 (28) 19 (23) 40 (49)  18 (22) 26 (32) 38 (46)  
Stage     0.45    0.61    0.77 
 1 21 8 (38) 8 (38) 5 (24)  8 (38) 4 (19) 9 (43)  8 (38) 4 (19) 9 (43)  
 2 103 24 (23) 33 (32) 46 (45)  32 (31) 32 (31) 39 (38)  29 (28) 31 (30) 43 (42)  
 3 41 11 (27) 12 (29) 18 (44)  10 (24) 11 (27) 20 (49)  10 (24) 13 (32) 18 (44)  
Grade     0.18    0.34    0.62 
 1 36 12 (33) 10 (28) 14 (39)  14 (39) 7 (19) 15 (42)  13 (36) 9 (25) 14 (39)  
 2 81 19 (24) 22 (27) 40 (49)  19 (24) 26 (32) 36 (44)  19 (23) 24 (30) 38 (47)  
 3 48 12 (25) 21 (44) 15 (31)  17 (35) 14 (29) 17 (35)  15 (31) 15 (31) 18 (38)  
Node     0.99*    0.46    0.69 
 0 57 14 (25) 20 (35) 23 (40)  15 (26) 17 (30) 25 (44)  17 (30) 13 (23) 27 (47)  
 >0 108 29 (27) 33 (30) 46 (43)  35 (32) 30 (28) 43 (40)  30 (28) 35 (32) 43 (40)  
Age (y)  54 (33–74) 50 (30–75) 48 (25–69) 0.32 51 (34–72) 51 (25–69) 48 (29–75) 0.89 49 (30–74) 49 (34–69) 52 (25–75) 0.99 
Tumor size (cm)  3 (0–11) 4 (1.9–8) 3 (1.9–8) 0.41 3 (1.9–8) 4 (1–8) 4 (1–8) 0.29 4 (1.9–8) 4 (1–8) 3 (1–8) 0.67 

NOTE. ErbB2 levels were measured against phosphorylated Akt, mTOR, and 4E-BP1 as well as clinicopathological variables. Results indicate that overexpression of ErbB2 correlates with increased phosphorylation levels of Akt, mTOR, and 4E-BP1, and p-Akt was found to positively correlate with ER status.

*

Cochran-Armitage trend test.

χ2 test.

Median (range), Wilcoxon test.

Table 3

Summary of survival according to tumor characteristics

FactornRecurrences5-year DFSP
Total 165 58 0.67  
ErbB2    <0.01 
 Low 112 31 0.77  
 High 53 27 0.44  
p-AKT    0.05 
 0 43 15 0.72  
 1 53 12 0.77  
 2 69 31 0.56  
mTOR    <0.01 
 0 50 0.87  
 1 47 20 0.66  
 2 68 30 0.52  
4E-BP1    0.05 
 0 47 13 0.76  
 1 48 12 0.72  
 2 70 33 0.57  
Age (y)    0.27 
 ≤50 99 38 0.62  
 >50 66 20 0.74  
Tumor size (cm)    0.20 
 <2 28 0.80  
 2–5 111 42 0.66  
 >5 26 10 0.56  
ER    0.32 
 Negative 83 32 0.62  
 Positive 82 26 0.71  
Stage    0.12 
 1 21 0.82  
 2 103 38 0.68  
 3 41 16 0.55  
Grade    0.01 
 1 36 0.87  
 2 81 30 0.67  
 3 48 21 0.47  
Node    0.03 
 0 57 14 0.78  
 >0 108 44 0.61  
FactornRecurrences5-year DFSP
Total 165 58 0.67  
ErbB2    <0.01 
 Low 112 31 0.77  
 High 53 27 0.44  
p-AKT    0.05 
 0 43 15 0.72  
 1 53 12 0.77  
 2 69 31 0.56  
mTOR    <0.01 
 0 50 0.87  
 1 47 20 0.66  
 2 68 30 0.52  
4E-BP1    0.05 
 0 47 13 0.76  
 1 48 12 0.72  
 2 70 33 0.57  
Age (y)    0.27 
 ≤50 99 38 0.62  
 >50 66 20 0.74  
Tumor size (cm)    0.20 
 <2 28 0.80  
 2–5 111 42 0.66  
 >5 26 10 0.56  
ER    0.32 
 Negative 83 32 0.62  
 Positive 82 26 0.71  
Stage    0.12 
 1 21 0.82  
 2 103 38 0.68  
 3 41 16 0.55  
Grade    0.01 
 1 36 0.87  
 2 81 30 0.67  
 3 48 21 0.47  
Node    0.03 
 0 57 14 0.78  
 >0 108 44 0.61  

NOTE. Univariate survival analysis to indicate DFS with phosphorylated Akt/mTOR/4E-BP1 and with other clinical factors. Phosphorylation of Akt, mTOR, and 4E-BP1 is statistically correlated with DFS.

Table 4

Association of p-p70S6K with p-mTOR, p-AKT, and 4-EBP1

MarkersTotalp-p70S6Kn (%)P                  *
012
Total 155 51 33 71  
p-mTOR     <0.001 
 0 47 28 (60) 14 (30) 5 (10)  
 1 47 14 (30) 6 (13) 27 (57)  
 2 61 9 (15) 13 (21) 39 (64)  
p-Akt     <0.001 
 0 41 29 (71) 3 (7) 9 (2)  
 1 49 11 (22) 19 (39) 19 (39)  
 2 65 11 (17) 11 (17) 43 (66)  
p-4E-BP1     <0.001 
 0 45 27 (60) 10 (22) 8 (18)  
 1 45 19 (42) 11 (24) 15 (34)  
 2 65 5 (8) 12 (18) 48 (74)  
MarkersTotalp-p70S6Kn (%)P                  *
012
Total 155 51 33 71  
p-mTOR     <0.001 
 0 47 28 (60) 14 (30) 5 (10)  
 1 47 14 (30) 6 (13) 27 (57)  
 2 61 9 (15) 13 (21) 39 (64)  
p-Akt     <0.001 
 0 41 29 (71) 3 (7) 9 (2)  
 1 49 11 (22) 19 (39) 19 (39)  
 2 65 11 (17) 11 (17) 43 (66)  
p-4E-BP1     <0.001 
 0 45 27 (60) 10 (22) 8 (18)  
 1 45 19 (42) 11 (24) 15 (34)  
 2 65 5 (8) 12 (18) 48 (74)  

NOTE. p-p70S6K was measured in tumor samples from 155 patients. Results indicate that p-p70S6K is statistically significantly and positively associated with p-mTOR, p-Akt, and p-4E-BP1.

*

χ2 test or Fisher’s exact test.

The authors would like to thank David Galloway for careful reading of the manuscript.

1
Beckmann MW, Niederacher D, Schnurch HG, Gusterson BA, Bender HG. Multistep carcinogenesis of breast cancer and tumour heterogeneity.
J Mol Med
1997
;
75
:
429
-39.
2
Borg A, Ferno M, Peterson C. Predicting the future of breast cancer.
Nat Med
2003
;
9
:
16
-8.
3
Blume-Jensen P, Hunter T. Oncogenic kinase signalling.
Nature (Lond)
2001
;
411
:
355
-65.
4
Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRAP/mTOR.
Genes Dev
2001
;
15
:
807
-26.
5
Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy.
Curr Opin Pharmacol
2003
;
3
:
371
-7.
6
Chen J, Fang Y. A novel pathway regulating the mammalian target of rapamycin (mTOR) signaling.
Biochem Pharmacol
2002
;
64
:
1071
-7.
7
Mita MM, Mita A, Rowinsky EK. The molecular target of rapamycin (mTOR) as a therapeutic target against cancer.
Cancer Biol Ther
2003
;
2
:
S169
-77.
8
Pyronnet S, Sonenberg N. Cell-cycle-dependent translational control.
Curr Opin Genet Dev
2001
;
11
:
13
-8.
9
Schmelzle T, Hall MN. TOR, a central controller of cell growth.
Cell
2000
;
103
:
253
-62.
10
Huang S, Houghton PJ. Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic.
Curr Opin Investig Drugs
2002
;
3
:
295
-304.
11
Mita MM, Mita A, Rowinsky EK. Mammalian target of rapamycin: a new molecular target for breast cancer.
Clin Breast Cancer
2003
;
4
:
126
-37.
12
Perez-Tenorio G, Stal O. Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients.
Br J Cancer
2002
;
86
:
540
-5.
13
Zhou BP, Hu MC, Miller SA, et al HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway.
J Biol Chem
2000
;
275
:
8027
-31.
14
Schmitz KJ, Otterbach F, Callies R, et al Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases.
Mod Pathol
2004
;
17
:
15
-21.
15
Yu D, Hung MC. Overexpression of ErbB2 in cancer and ErbB2-targeting strategies.
Oncogene
2000
;
19
:
6115
-21.
16
Zhou BP, Liao Y, Xia W, et al HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.
Nat Cell Biol
2001
;
3
:
973
-82.
17
Stoica GE, Franke TF, Wellstein A, et al Heregulin-beta1 regulates the estrogen receptor-alpha gene expression and activity via the ErbB2/PI 3-K/Akt pathway.
Oncogene
2003
;
22
:
2073
-87.
18
Stoica GE, Franke TF, Moroni M, et al Effect of estradiol on estrogen receptor-alpha gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway.
Oncogene
2003
;
22
:
7998
-8011.
19
Campbell RA, Bhat-Nakshatri P, Patel NM, et al Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance.
J Biol Chem
2001
;
276
:
9817
-24.
20
Yu K, Toral-Barza L, Discafani C, et al mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer.
Endocr Relat Cancer
2001
;
8
:
249
-58.
21
Sellappan S, Grijalva R, Zhou X, et al Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line.
Cancer Res
2004
;
64
:
3479
-85.
22
Yang W, Klos KS, Zhou X, et al ErbB2 overexpression in human breast carcinoma is correlated with p21Cip1 up-regulation and tyrosine-15 hyperphosphorylation of p34Cdc2: poor responsiveness to chemotherapy with cyclophoshamide methotrexate, and 5-fluorouracil is associated with Erb2 overexpression and with p21Cip1 overexpression.
Cancer (Phila)
2003
;
98
:
1123
-30.
23
Yu D, Liu B, Tan M, et al Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to Taxol via mdr-1-independent mechanisms.
Oncogene
1996
;
13
:
1359
-65.
24
Benz CC, Scott GK, Sarup JC, et al Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu.
Breast Cancer Res Treat
1993
;
24
:
85
-95.
25
Tan M, Jing T, Lan KH, et al Phosphorylation on tyrosine-15 of p34Cdc2 by ErbB2 inhibits p34Cdc2 activation and is involved in resistance to Taxol-induced apoptosis.
Mol Cell
2002
;
9
:
993
-1004.
26
Tan M, Yao J, Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities.
Cancer Res
1997
;
57
:
1199
-205.
27
Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy.
Cell Signal
2002
;
14
:
381
-95.
28
Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer.
Nat Rev Cancer
2002
;
2
:
489
-501.
29
Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation.
Biochem J
1999
;
344
:
427
-31.
30
Sekulic A, Hudson CC, Homme JL, et al A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.
Cancer Res
2000
;
60
:
3504
-13.
31
Scott PH, Lawrence JC, Jr. Attenuation of mammalian target of rapamycin activity by increased cAMP in 3T3–L1 adipocytes.
J Biol Chem
1998
;
273
:
34496
-501.
32
Reynolds THt, Bodine SC, Lawrence JC, Jr. Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load.
J Biol Chem
2002
;
277
:
17657
-62.
33
Brunn GJ, Hudson CC, Sekulic A, et al Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.
Science (Wash DC)
1997
;
277
:
99
-101.
34
Yu D, Jing T, Liu B, et al Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase.
Mol Cell
1998
;
2
:
581
-91.
35
Simoncini T, Hafezi-Moghadam A, Brazil DP, et al Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase.
Nature (Lond)
2000
;
407
:
538
-41.
36
Zhou BP, Hung MC. Dysregulation of cellular signaling by HER2/neu in breast cancer.
Semin Oncol
2003
;
30
:
38
-48.
37
Esteva FJ, Sahin AA, Cristofanilli M, Arun B, Hortobagyi GN. Molecular prognostic factors for breast cancer metastasis and survival.
Semin Radiat Oncol
2002
;
12
:
319
-28.
38
Gago FE, Tello OM, Diblasi AM, Ciocca DR. Integration of estrogen and progesterone receptors with pathological and molecular prognostic factors in breast cancer patients.
J Steroid Biochem Mol Biol
1998
;
67
:
431
-7.
39
Harding MW. Immunophilins, mTOR, and pharmacodynamic strategies for a targeted cancer therapy.
Clin Cancer Res
2003
;
9
:
2882
-6.
40
Raught B, Gingras AC, Sonenberg N. The target of rapamycin (TOR) proteins.
Proc Natl Acad Sci USA
2001
;
98
:
7037
-44.
41
Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling.
J Biol Chem
2002
;
277
:
23977
-80.
42
Neshat MS, Mellinghoff IK, Tran C, et al Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR.
Proc Natl Acad Sci USA
2001
;
98
:
10314
-9.
43
Shi Y, Gera J, Hu L, et al Enhanced sensitivity of multiple myeloma cells containing PTEN mutations to CCI-779.
Cancer Res
2002
;
62
:
5027
-34.
44
Fairclough DL, Gagnon DD, Zagari MJ, Marschner N, Dicato M. Evaluation of quality of life in a clinical trial with nonrandom dropout: the effect of epoetin alpha in anemic cancer patients.
Qual Life Res
2003
;
12
:
1013
-27.