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Molecular Oncology, Markers, Clinical Correlates |
Departments of 1 Surgical Oncology and 2 Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and 3 Department of Pathology, Cancer Hospital, Fudan University, Shanghai, People’s Republic of China
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 ß-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 x 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 (x200) 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 
2 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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B 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 2 analysis (P < 0.01; data not shown), which is consistent with in vitro studies on the causal association of the molecules (6
, 7)
.
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; 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.
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. 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)
.
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. 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. 4A)
.
|
. There was no significant change in p-mTOR after rapamycin treatment, which is consistent with the function of rapamycin (Fig. 4A)
. 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. 4B)
. 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. 4C)
. 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. 4C
), 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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 (2 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. 4C)
. 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. 4C)
. 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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{at}mdanderson.org
Received 1/19/04; revised 7/ 1/04; accepted 7/13/04.
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P. Bali, M. Pranpat, R. Swaby, W. Fiskus, H. Yamaguchi, M. Balasis, K. Rocha, H.-G. Wang, V. Richon, and K. Bhalla Activity of Suberoylanilide Hydroxamic Acid Against Human Breast Cancer Cells with Amplification of Her-2 Clin. Cancer Res., September 1, 2005; 11(17): 6382 - 6389. |
