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mTOR in breast cancer: Differential expression in triple-negative and non-triple-negative tumors
The Breast 21 (2012) 178e182
Contents lists available at SciVerse ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
mTOR in breast cancer: Differential expression in triple-negative and non-triple-negative tumors S. Walsha, L. Flanaganb, C. Quinnb, D. Evoya, E.W. McDermotta, A. Piercea, M.J. Duffya, b, * a b
UCD School of Medicine and Medical Science, Conway Institute, University College Dublin, Dublin, Ireland Department of Pathology and Laboratory Medicine, St Vincent’s University Hospital, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2010 Received in revised form 12 July 2011 Accepted 2 September 2011
Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptors (ER), progesterone receptors (PR) and overexpression of HER2. Targeted therapy is currently unavailable for this subgroup of breast cancer patients. mTOR controls cancer cell growth, survival and invasion and is thus a potential target for the treatment of patients with TNBC. Using immunohistochemistry, mTOR and p-mTOR were measured in 89 TNBCs and 99 non-TNBCs. While mTOR expression was confined to tumor cell cytoplasm, p-mTOR staining was located in the nucleus, perinuclear area and in the cytoplasm. Potentially important, was our finding that nuclear p-mTOR was found more frequently in triple-negative than non triplenegative cancers (p < 0.001). These results suggest that mTOR may play a more important role in the progression of TNBC compared to non-TNBC. Based on these findings, we conclude that mTOR may be a new target for the treatment of triple-negative breast cancer. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Breast cancer Triple-negative mTOR Rapamycin
Introduction Targeted therapy is currently available for the majority of patients with newly diagnosed breast cancer. Thus, patients with estrogen receptor (ER)-positive and/or progesterone receptor (PR)positive disease receive hormone therapy, while patients with HER2-positive disease may be treated with trastuzumab (Herceptin). For patients lacking ER/PR and HER2 overexpression however, targeted therapy is currently unavailable.1 Consequently, the only systemic therapy available for this subgroup of patients which have been dubbed triple-negative is chemotherapy.2 Currently, there is intense interest in developing new targeted therapies for patients with triple-negative breast cancer. The growth, invasion and metastasis of cancer are driven by a series of intracellular cell signalling pathways.3 One of the beststudied pathways is the PI3K system which transmits signals from a number of receptors including EGFR, HER2 and HER3.4,5 This pathway is frequently activated in cancer, including breast cancer.5,6 A key downstream component in the PI3K pathway is the serine/threonine kinase known as mTOR. mTOR is thus a critical regulator of tumor formation and progression. Consistent with its role in tumor formation and progression, targeted therapy against
* Corresponding author. Department of Pathology and Laboratory Medicine, St Vincent’s University Hospital, Dublin 4, Ireland. Tel.: þ353 1 2214607. E-mail address: [email protected] (M.J. Duffy). 0960-9776/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2011.09.008
mTOR has been shown to decrease tumor growth in model systems.7e9 Indeed, several agents that inhibit mTOR are currently in clinical trials for the treatment of multiple cancer types,5,6 including breast cancer.6 Three mTOR antagonists are currently undergoing evaluation in breast cancer, everolimus, deforolimus and temsirolimus.5 The aim of this study was to investigate the expression of mTOR and its putative activated form, phospho mTOR (p-mTOR), in breast cancer. In particular, we wished to establish if these proteins were expressed in patients with triple-negative disease. If expressed in this subgroup of patients, mTOR or more importantly its activated form, p-mTOR, could be a potential target for treating patients with breast cancer that is negative for ER, PR and HER2. Materials and methods Patient selection The patients were diagnosed over the period 2000e2006 and were selected from a total population of approximately 1500 new cases of breast cancer. Initially, 100 TN samples were selected from this cohort. Of these 100 however, 89 were suitable for immunostaining in the tissue microarray slides stained for mTor and 88 were suitable for immunostaining in those stained for p-mTor. Unsuitability was due to loss of sample in preparing TMA or lack of tumor cells in the section examined. As a control population, 100
S. Walsh et al. / The Breast 21 (2012) 178e182
non-TN samples were randomly selected. Of these, 99 were suitable for analysis. Pathological and biochemical characteristics of the TN and non-TN tumors are summarized in Table 1. ER, PR and HER2 status were determined using standard immunohistochemistry. The antibodies used were monoclonal rabbit estrogen receptor antibody (SP1, Thermo Scientific), monoclonal mouse anti-human progesterone receptor (clone PgR 636, Dako Cytomation) and anti-Her-2/neu rabbit monoclonal antibody (4B5, Ventana Medical Solutions Inc). ER and PR positivity were defined as 10% of cells staining positively. HER2 positivity was defined as uniform intense staining in >30% of invasive tumor cells. Patient age and characteristics of the tumors investigated are summarized in Table 1. Tumor grade was determined by the modified Bloom and Richardson method taking account of tubule formation, nuclear pleomorphism and mitotic activity.10 Immunohistochemistry Tissue microarrays were constructed and prepared for immunohistochemistry as previously described.11 Staining for mTOR and p-mTOR were carried out using rabbit monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA, mTor 7C10, Rabbit mAb #2983 and p-mTor-Ser2448, 49F9, Rabbit mAb #2976). The mTOR and p-mTOR antibodies were applied to the slides at concentrations of 1:50 and 1:200, respectively. After overnight incubation at 4 C, sections were washed in phosphate bufferedTween (PBS-Tween) (0.1%). They were then incubated for 30 min with diluted biotinylated secondary antibody solution (Vectastain Elite ABC kit). Following a 5 min PBS wash, the slides were incubated with Vectastain Elite ABC reagent. Sections of prostate adenocarcinoma were used as positive controls for both mTOR and p-mTOR. Negative controls were incubated either with PBS or with the primary antibodies combined with excess blocking peptides (mTOR blocking peptide #1072 and p-mTOR blocking peptide Ser2448, #1230, Cell Signaling Technology). The ratio of blocking peptide to antibody was 5:1. Staining was evaluated and scored by 2 independent investigators. Discrepancies were resolved to achieve concordance. As there is no established cut-off point for mTOR
Table 1 Relationship between tumor characteristics and triple-negative/non triple-negative status. LN, lymph node; LVI, lymphovascular invasion; NS, non significant. Triple negative n (%) Age at Diagnosis (yr) 50 35 (39%) >50 54 (61%) Tumor Grade Grade 1 0 Grade 2 13 (15%) Grade 3 76 (85%) Histological Type Ductal 83 (93%) Lobular 5 (5.6%) Other 0 Unknown 1 (1%) Tumor Size (cm) 2 48 (54%) >2 39 (44%) Unknown 2 (2%) LN Status Negative 50 (56%) Positive 37 (41%) Unknown 2 (2%) LVI status Negative 44 (50%) Positive 44 (50%) Unknown 1 (1.1%)
Non-triple negative n (%)
p-value 0.015
23 (23%) 77 (77%) <0.0001 14 (14%) 61 (61%) 25 (25%) <0.0001 69 (69%) 24 (24%) 7 (7%) 0 NS 51 (51%) 49 (49%) 0 NS 56 (56%) 40 (40%) 3 (3%) NS 37 (37%) 60 (60%) 3 (3%)
179
immunostaining, we investigated a series of cut-off points, ie, 10%, 20%, 50% tumor cell staining. Irrespective of the cut-off point chosen, similar conclusions emerged. We decided to use 20% cut-off point in this article in order to be consistent with our previous report on Src using these samples. The Mann-Whiney U test and chi square tests (SPSS version 15.0) were used to assess statistical significance. P values <0.05 were considered statistically significant. Results Characteristics of triple-negative and non triple-negative breast cancers Compared with non triple-negative breast tissues, triplenegative cancers were more frequent in patients diagnosed at 50 years of age than in those diagnosed at >50 years; in high grade (grade 3) rather than in low grade (grade 1 and 2) tumors; and in ductal compared with lobular cancers (Table 1). In contrast, triple negativity was unrelated to tumor size, lymph node status or lymphovascular invasion. Expression of mTOR and p-mTOR in total population of samples mTOR was located exclusively in the cytoplasm and was present in 88 (47%) of the 188 samples investigated. P-mTOR however, was detected not only in the cytoplasm (39% of samples), but also in the perinuclear area (21.9% of samples) and in the nucleus (22.5% of samples). Typical staining patterns for mTOR and p-mTOR are shown in Fig. 1. Relationship between mTOR and p-mTOR and tumor characteristics Table 2 summarises the relationship between both mTOR and pmTOR and tumor characteristics. mTOR was more frequently detected in high grade (grade 3) than in low grade (grade I and II) cancers (p ¼ 0.009). mTOR was unrelated to tumor size, lymph node status, vascular invasion or age of patient at diagnosis. Expression of nuclear and cytoplasmic p-mTOR in the total population was independent of all the clinicopathological factors investigated. However, perinuclear p-mTOR was detected more frequently in low grade than high grade tumors (p < 0.001) The perinuclear form of mTOR was present more frequently in nontriple negative tumours from patients diagnosed at under 50 years than those who were over fifty years old at diagnosis (p ¼ 0.002) and in triple-negative tumors which were lymph node positive (p ¼ 0.042). Finally, cytoplasmic p-mTOR was detected more frequently in ductal than in lobular tumors although this significant difference was confined to the non-triple-negative subgroup (p ¼ 0.03). Comparative expression of mTOR and p-mTOR in triple-negative and non triple-negative breast cancers mTOR was expressed in a similar proportion of patients with triple-negative and non triple-negative breast cancer (53% vs 41%; p, non-significant). As can be seen in Table 3 however, nuclear p-mTOR was detected more frequently in triple-negative than non triple-negative samples (p < 0.001). In contrast, perinuclear staining was found more frequently in the non triple-negative than in triple-negative samples (p < 0.001). Cytoplasmic p-mTOR was found in a similar proportion of triple-negative and non triplenegative samples (37.5% vs 39%).
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S. Walsh et al. / The Breast 21 (2012) 178e182
Discussion Rationally, it might be expected that prior to a new biological agent entering clinical trials, expression of its target would have undergone detailed study. Although mTOR is currently undergoing active investigation as a potential target for cancer treatment, few in-depth studies have been carried out on its expression in human cancers.7,10,12,13 In this, we found that high expression of both mTOR and it’s active form p-mTOR were present in almost half of the samples investigated. mTOR was present exclusively located in the cytoplasm, while p-mTOR was present in the nucleus, the perinuclear area and in the cytoplasm. Expression of both the active and inactive forms of mTOR was largely independent of the clinicopathological factors investigated. In the combined analysis of triple negative and non-triple negative
cancers, mTOR however, was expressed more frequently in high grade compared with low grade malignancies, in comparison with perinuclear p-mTOR, which was present more frequently in low grade breast carcinomas. Cytoplasmic p-mTOR was detected more frequently in ductal compared to lobular cancer, but this relationship was limited to the non-triple-negative patients. Two reports have previously been published on the expression of mTOR in breast cancer, both of which focused exclusively on the p-mTOR form.12,13 Unlike our study, neither of these differentiated between the different subcellular locations of p-mTOR. Similarly, neither compared expression in triple negative and non-triple negative breast cancers. Similar to our findings, Bose et al12 found increased levels of p-mTOR in high grade versus low grade cancers. However, Zhou et al13 found no relationship between p-mTOR and tumor grade. These different findings in relation to tumor grade
Fig. 1. (A) Cytoplasmic immunostaining of mTOR. (B) Negative control for mTOR, antibody with blocking peptide. (C) Nuclear immunostaining of p-mTOR (D) Perinuclear immunostaining of p-mTOR (E) Cytoplasmic immunostaining of p-mTOR (F) Negative control, p-mTOR, antibody with blocking peptide. (Magnification 20).
S. Walsh et al. / The Breast 21 (2012) 178e182
181
Table 2 Relationship between mTOR and p-mTOR and tumor characteristics. PN, perinuclear; LVI, lymphovascular invasion; N/S, not significant. Clinicopathological Characteristics
Size (cm) 2 (n ¼ 99) >2 (n ¼ 88) Unknown (n ¼ 2) Lymph node Status Negative (n ¼ 106) Positive (n ¼ 77) Unknown (n ¼ 5) Tumor Grade 1 and 2 (n ¼ 80) 3 (n ¼ 102) LVI Negative (n ¼ 81) Positive (n ¼ 104) Unknown (n ¼ 4) Histological Type Ductal (n ¼ 152) Lobular (n ¼ 29) Other (n ¼ 7) Unknown (n ¼ 1) Age at Diagnosis (yr) 50 (n ¼ 58) >50 (n ¼ 131)
Positive for mTOR
Positive for Cyto p-mTOR
Positive for PN p-mTOR
Positive for Nuclear p-mTOR
n (%)
p-value
n (%)
p-value
n (%)
p-value
n (%)
48 (48%) 39 (44%) 1 (50%)
N/S
40 (40%) 31 (35%) 1 (50%)
N/S
21 (21%) 20 (23%) 0
N/S
24 (24%) 17 (19%) 1 (50%)
55 (52%) 32 (42%) 1 (20%)
N/S
41 (39%) 28 (36%) 3 (60%)
N/S
24 (23%) 13 (17%) 4 (80%)
N/S
22 (21%) 19 (25%) 1 (20%)
32 (40%) 56 (55%)
0.009
33 (41%) 39 (38%)
N/S
31 (39%) 10 (10%)
<0.001
16 (20%) 26 (25%)
41 (51%) 46 (44%) 1 (25%)
N/S
32 (40%) 38 (37%) 2 (50%)
N/S
17 (21%) 23 (22%) 1 (25%)
N/S
18 (22%) 22 (21%) 2 (50%)
73 (48%) 10 (34%) 5 (71%) 0
N/S
62 (41%) 8 (28%) 2 (29%) 0
N/S
28 (18%) 11 (38%) 2 (29%) 0
N/S
37 (24%) 5 (17%) 0 0
28 (48%) 60 (46%)
N/S
22 (38%) 50 (38%)
N/S
16 (28%) 25 (19%)
N/S
12 (21%) 30 (23%)
p-value N/S
N/S
N/S
N/S
N/S
N/S
may possibly be attributed to the lack of reproducibility in determining this tumor characteristic. A potentially important finding in this study was that the putative active form of mTOR, i.e., p-mTOR, was differentially expressed in triple negative and non-triple negative cancers. In particular, nuclear p-mTOR was expressed more frequently in the triple-negative than non-triple negative samples. In a previous study using ovarian granulosa cells in culture, Yaba et al14 reported that p-mTOR correlated with the active status of mTOR. Using breast cancer cell lines in culture, Vazquez-Martin found that that the nuclear accumulation of a specific form of p-mTOR closely correlated with proliferative capacity.15 Furthermore, mTOR was mostly cytoplasmic and perinuclear in non-dividing cells but was tightly localised to the mitotic spindle in M-phase cells. These studies therefore suggest that the presence of p-mTOR corresponds with the activation of mTor and an increase in proliferation. In a further study, Tsang et al16 found that nuclear localized mTOR mediated transcription of rDNA and tRNA, nucleic acids that are critical for protein synthesis. These findings when taken together suggest that nuclear mTOR is the main biological form of this protein. Our results thus suggest that active nuclear mTOR plays a more important role in the development and/or progression of triple-negative than non-triple negative breast cancers. Consequently, triple-negative tumours may be more sensitive than non triple-negative tumors to treatment with inhibitors that target mTOR. In conclusion, our finding of high expression of nuclear p-mTOR in approximately 36% of triple-negative breast carcinomas suggests
Table 3 Comparative expression p-mTOR in triple negative (TN) and non-TN breast cancers. NS, not significant; PN, perinuclear. Status
No. of patients
Positive for nuclear p-mTOR n (%)
Positive for PN p-mTOR n (%)
Positive for cytoplasmic p-mTOR n (%)
TN Non TN p-value
88 99
32 (36) 10 (10) <0.001
4 (4) 37 (37) <0.001
33 (37.5) 39 (39) NS
that inhibitors directed against this protein may be effective in at least some patients with this subtype of breast cancer. Patients likely to respond may be those expressing high levels of mTOR/pmTOR. This hypothesis should now be considered for testing in clinical trials. Conflict of interest statement The authors have no conflicts of interest to declare. Ethical approval Ethical approval for this study was obtained from the St, Vincent’s University Hospital Ethics Committee. Acknowledgement The authors wish to thank the Science Foundation Ireland, Strategic Research Cluster award (08/SRC/B1410) to Molecular Therapeutics for Cancer Ireland for funding this work. The sponsors had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript or the decision to submit the manuscript for publication. References 1. Podo F, Buydens LMC, Degani H, Buydens LM, Degani H, Hilhorst R, et al. Triple-negative breast cancer: present challenges and new perspectives. Mol Oncol. 4(3): p. 209e229. 2. Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N. Triple-negative breast cancerecurrent status and future directions. Ann Oncol 2009;20(12):1913e27. 3. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006;441(7092):424e30. 4. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci 2009;122(Pt 20):3589e94. 5. Courtney KD, Corcoran RB, Engelman JA. The PI3K pathway as drug target in human cancer. J Clin Oncol. 28(6): pp. 1075e1083. 6. Alvarez RH, Valero V, Hortobagyi GN. Emerging targeted therapies for breast cancer. J Clin Oncol. 28(20): pp. 3366e3379. 7. Mabuchi S, Kawase C, Altomare DA, Morishige K, Sawada K, Hayashi M, et al. mTOR is a promising therapeutic target both in cisplatin-sensitive and
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S. Walsh et al. / The Breast 21 (2012) 178e182 cisplatin-resistant clear cell carcinoma of the ovary. Clin Cancer Res 2009;15(17):5404e13. Lu CH, Wyszomierski SL, Tseng LM, Sun MH, Lan KH, Neal CL, et al. Preclinical testing of clinically applicable strategies for overcoming trastuzumab resistance caused by PTEN deficiency. Clin Cancer Res 2007;13(19):5883e8. Chresta CM, Davies BR, Hickson I, Harding T, Cosulich S, Critchlow SE, et al. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res 70(1): p. 288e298. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with longterm follow-up. Histopathology 2002;41(3A):154e61. Tryfonopoulos D, Walsh S, Collins DM, Flanagan L, Quinn C, Corkery B, et al. Src: a potential target for the treatment of triple-negative breast cancer. Ann Oncol, in press.
12. Bose S, Chandran S, Mirocha JM, Bose N. The Akt pathway in human breast cancer: a tissue-array-based analysis. Mod Pathol 2006;19(2):238e45. 13. Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, et al. Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res 2004;10(20):6779e88. 14. Yaba A, Bianchi V, Borini A, Johnson J. A putative mitotic checkpoint dependent on mTOR function controls cell proliferation and survival in ovarian granulosa cells. Reprod Sci 2008;15(2):128e38. 15. Vazquez-Martin A, Oliveras-Ferraros C, Bernadó L, López-Bonet E, Menendez JA. The serine 2481-autophosphorylated form of mammalian Target Of Rapamycin (mTOR) is localized to midzone and midbody in dividing cancer cells. Biochem Biophys Res Commun 2009;380(3):638e43. 16. Tsang CK, Liu H, Zheng XF. mTOR binds to the promoters of RNA polymerase Iand III-transcribed genes. Cell Cycle. 9(5): pp. 953e957.
Contents lists available at SciVerse ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
mTOR in breast cancer: Differential expression in triple-negative and non-triple-negative tumors S. Walsha, L. Flanaganb, C. Quinnb, D. Evoya, E.W. McDermotta, A. Piercea, M.J. Duffya, b, * a b
UCD School of Medicine and Medical Science, Conway Institute, University College Dublin, Dublin, Ireland Department of Pathology and Laboratory Medicine, St Vincent’s University Hospital, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2010 Received in revised form 12 July 2011 Accepted 2 September 2011
Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptors (ER), progesterone receptors (PR) and overexpression of HER2. Targeted therapy is currently unavailable for this subgroup of breast cancer patients. mTOR controls cancer cell growth, survival and invasion and is thus a potential target for the treatment of patients with TNBC. Using immunohistochemistry, mTOR and p-mTOR were measured in 89 TNBCs and 99 non-TNBCs. While mTOR expression was confined to tumor cell cytoplasm, p-mTOR staining was located in the nucleus, perinuclear area and in the cytoplasm. Potentially important, was our finding that nuclear p-mTOR was found more frequently in triple-negative than non triplenegative cancers (p < 0.001). These results suggest that mTOR may play a more important role in the progression of TNBC compared to non-TNBC. Based on these findings, we conclude that mTOR may be a new target for the treatment of triple-negative breast cancer. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Breast cancer Triple-negative mTOR Rapamycin
Introduction Targeted therapy is currently available for the majority of patients with newly diagnosed breast cancer. Thus, patients with estrogen receptor (ER)-positive and/or progesterone receptor (PR)positive disease receive hormone therapy, while patients with HER2-positive disease may be treated with trastuzumab (Herceptin). For patients lacking ER/PR and HER2 overexpression however, targeted therapy is currently unavailable.1 Consequently, the only systemic therapy available for this subgroup of patients which have been dubbed triple-negative is chemotherapy.2 Currently, there is intense interest in developing new targeted therapies for patients with triple-negative breast cancer. The growth, invasion and metastasis of cancer are driven by a series of intracellular cell signalling pathways.3 One of the beststudied pathways is the PI3K system which transmits signals from a number of receptors including EGFR, HER2 and HER3.4,5 This pathway is frequently activated in cancer, including breast cancer.5,6 A key downstream component in the PI3K pathway is the serine/threonine kinase known as mTOR. mTOR is thus a critical regulator of tumor formation and progression. Consistent with its role in tumor formation and progression, targeted therapy against
* Corresponding author. Department of Pathology and Laboratory Medicine, St Vincent’s University Hospital, Dublin 4, Ireland. Tel.: þ353 1 2214607. E-mail address: [email protected] (M.J. Duffy). 0960-9776/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2011.09.008
mTOR has been shown to decrease tumor growth in model systems.7e9 Indeed, several agents that inhibit mTOR are currently in clinical trials for the treatment of multiple cancer types,5,6 including breast cancer.6 Three mTOR antagonists are currently undergoing evaluation in breast cancer, everolimus, deforolimus and temsirolimus.5 The aim of this study was to investigate the expression of mTOR and its putative activated form, phospho mTOR (p-mTOR), in breast cancer. In particular, we wished to establish if these proteins were expressed in patients with triple-negative disease. If expressed in this subgroup of patients, mTOR or more importantly its activated form, p-mTOR, could be a potential target for treating patients with breast cancer that is negative for ER, PR and HER2. Materials and methods Patient selection The patients were diagnosed over the period 2000e2006 and were selected from a total population of approximately 1500 new cases of breast cancer. Initially, 100 TN samples were selected from this cohort. Of these 100 however, 89 were suitable for immunostaining in the tissue microarray slides stained for mTor and 88 were suitable for immunostaining in those stained for p-mTor. Unsuitability was due to loss of sample in preparing TMA or lack of tumor cells in the section examined. As a control population, 100
S. Walsh et al. / The Breast 21 (2012) 178e182
non-TN samples were randomly selected. Of these, 99 were suitable for analysis. Pathological and biochemical characteristics of the TN and non-TN tumors are summarized in Table 1. ER, PR and HER2 status were determined using standard immunohistochemistry. The antibodies used were monoclonal rabbit estrogen receptor antibody (SP1, Thermo Scientific), monoclonal mouse anti-human progesterone receptor (clone PgR 636, Dako Cytomation) and anti-Her-2/neu rabbit monoclonal antibody (4B5, Ventana Medical Solutions Inc). ER and PR positivity were defined as 10% of cells staining positively. HER2 positivity was defined as uniform intense staining in >30% of invasive tumor cells. Patient age and characteristics of the tumors investigated are summarized in Table 1. Tumor grade was determined by the modified Bloom and Richardson method taking account of tubule formation, nuclear pleomorphism and mitotic activity.10 Immunohistochemistry Tissue microarrays were constructed and prepared for immunohistochemistry as previously described.11 Staining for mTOR and p-mTOR were carried out using rabbit monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA, mTor 7C10, Rabbit mAb #2983 and p-mTor-Ser2448, 49F9, Rabbit mAb #2976). The mTOR and p-mTOR antibodies were applied to the slides at concentrations of 1:50 and 1:200, respectively. After overnight incubation at 4 C, sections were washed in phosphate bufferedTween (PBS-Tween) (0.1%). They were then incubated for 30 min with diluted biotinylated secondary antibody solution (Vectastain Elite ABC kit). Following a 5 min PBS wash, the slides were incubated with Vectastain Elite ABC reagent. Sections of prostate adenocarcinoma were used as positive controls for both mTOR and p-mTOR. Negative controls were incubated either with PBS or with the primary antibodies combined with excess blocking peptides (mTOR blocking peptide #1072 and p-mTOR blocking peptide Ser2448, #1230, Cell Signaling Technology). The ratio of blocking peptide to antibody was 5:1. Staining was evaluated and scored by 2 independent investigators. Discrepancies were resolved to achieve concordance. As there is no established cut-off point for mTOR
Table 1 Relationship between tumor characteristics and triple-negative/non triple-negative status. LN, lymph node; LVI, lymphovascular invasion; NS, non significant. Triple negative n (%) Age at Diagnosis (yr) 50 35 (39%) >50 54 (61%) Tumor Grade Grade 1 0 Grade 2 13 (15%) Grade 3 76 (85%) Histological Type Ductal 83 (93%) Lobular 5 (5.6%) Other 0 Unknown 1 (1%) Tumor Size (cm) 2 48 (54%) >2 39 (44%) Unknown 2 (2%) LN Status Negative 50 (56%) Positive 37 (41%) Unknown 2 (2%) LVI status Negative 44 (50%) Positive 44 (50%) Unknown 1 (1.1%)
Non-triple negative n (%)
p-value 0.015
23 (23%) 77 (77%) <0.0001 14 (14%) 61 (61%) 25 (25%) <0.0001 69 (69%) 24 (24%) 7 (7%) 0 NS 51 (51%) 49 (49%) 0 NS 56 (56%) 40 (40%) 3 (3%) NS 37 (37%) 60 (60%) 3 (3%)
179
immunostaining, we investigated a series of cut-off points, ie, 10%, 20%, 50% tumor cell staining. Irrespective of the cut-off point chosen, similar conclusions emerged. We decided to use 20% cut-off point in this article in order to be consistent with our previous report on Src using these samples. The Mann-Whiney U test and chi square tests (SPSS version 15.0) were used to assess statistical significance. P values <0.05 were considered statistically significant. Results Characteristics of triple-negative and non triple-negative breast cancers Compared with non triple-negative breast tissues, triplenegative cancers were more frequent in patients diagnosed at 50 years of age than in those diagnosed at >50 years; in high grade (grade 3) rather than in low grade (grade 1 and 2) tumors; and in ductal compared with lobular cancers (Table 1). In contrast, triple negativity was unrelated to tumor size, lymph node status or lymphovascular invasion. Expression of mTOR and p-mTOR in total population of samples mTOR was located exclusively in the cytoplasm and was present in 88 (47%) of the 188 samples investigated. P-mTOR however, was detected not only in the cytoplasm (39% of samples), but also in the perinuclear area (21.9% of samples) and in the nucleus (22.5% of samples). Typical staining patterns for mTOR and p-mTOR are shown in Fig. 1. Relationship between mTOR and p-mTOR and tumor characteristics Table 2 summarises the relationship between both mTOR and pmTOR and tumor characteristics. mTOR was more frequently detected in high grade (grade 3) than in low grade (grade I and II) cancers (p ¼ 0.009). mTOR was unrelated to tumor size, lymph node status, vascular invasion or age of patient at diagnosis. Expression of nuclear and cytoplasmic p-mTOR in the total population was independent of all the clinicopathological factors investigated. However, perinuclear p-mTOR was detected more frequently in low grade than high grade tumors (p < 0.001) The perinuclear form of mTOR was present more frequently in nontriple negative tumours from patients diagnosed at under 50 years than those who were over fifty years old at diagnosis (p ¼ 0.002) and in triple-negative tumors which were lymph node positive (p ¼ 0.042). Finally, cytoplasmic p-mTOR was detected more frequently in ductal than in lobular tumors although this significant difference was confined to the non-triple-negative subgroup (p ¼ 0.03). Comparative expression of mTOR and p-mTOR in triple-negative and non triple-negative breast cancers mTOR was expressed in a similar proportion of patients with triple-negative and non triple-negative breast cancer (53% vs 41%; p, non-significant). As can be seen in Table 3 however, nuclear p-mTOR was detected more frequently in triple-negative than non triple-negative samples (p < 0.001). In contrast, perinuclear staining was found more frequently in the non triple-negative than in triple-negative samples (p < 0.001). Cytoplasmic p-mTOR was found in a similar proportion of triple-negative and non triplenegative samples (37.5% vs 39%).
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S. Walsh et al. / The Breast 21 (2012) 178e182
Discussion Rationally, it might be expected that prior to a new biological agent entering clinical trials, expression of its target would have undergone detailed study. Although mTOR is currently undergoing active investigation as a potential target for cancer treatment, few in-depth studies have been carried out on its expression in human cancers.7,10,12,13 In this, we found that high expression of both mTOR and it’s active form p-mTOR were present in almost half of the samples investigated. mTOR was present exclusively located in the cytoplasm, while p-mTOR was present in the nucleus, the perinuclear area and in the cytoplasm. Expression of both the active and inactive forms of mTOR was largely independent of the clinicopathological factors investigated. In the combined analysis of triple negative and non-triple negative
cancers, mTOR however, was expressed more frequently in high grade compared with low grade malignancies, in comparison with perinuclear p-mTOR, which was present more frequently in low grade breast carcinomas. Cytoplasmic p-mTOR was detected more frequently in ductal compared to lobular cancer, but this relationship was limited to the non-triple-negative patients. Two reports have previously been published on the expression of mTOR in breast cancer, both of which focused exclusively on the p-mTOR form.12,13 Unlike our study, neither of these differentiated between the different subcellular locations of p-mTOR. Similarly, neither compared expression in triple negative and non-triple negative breast cancers. Similar to our findings, Bose et al12 found increased levels of p-mTOR in high grade versus low grade cancers. However, Zhou et al13 found no relationship between p-mTOR and tumor grade. These different findings in relation to tumor grade
Fig. 1. (A) Cytoplasmic immunostaining of mTOR. (B) Negative control for mTOR, antibody with blocking peptide. (C) Nuclear immunostaining of p-mTOR (D) Perinuclear immunostaining of p-mTOR (E) Cytoplasmic immunostaining of p-mTOR (F) Negative control, p-mTOR, antibody with blocking peptide. (Magnification 20).
S. Walsh et al. / The Breast 21 (2012) 178e182
181
Table 2 Relationship between mTOR and p-mTOR and tumor characteristics. PN, perinuclear; LVI, lymphovascular invasion; N/S, not significant. Clinicopathological Characteristics
Size (cm) 2 (n ¼ 99) >2 (n ¼ 88) Unknown (n ¼ 2) Lymph node Status Negative (n ¼ 106) Positive (n ¼ 77) Unknown (n ¼ 5) Tumor Grade 1 and 2 (n ¼ 80) 3 (n ¼ 102) LVI Negative (n ¼ 81) Positive (n ¼ 104) Unknown (n ¼ 4) Histological Type Ductal (n ¼ 152) Lobular (n ¼ 29) Other (n ¼ 7) Unknown (n ¼ 1) Age at Diagnosis (yr) 50 (n ¼ 58) >50 (n ¼ 131)
Positive for mTOR
Positive for Cyto p-mTOR
Positive for PN p-mTOR
Positive for Nuclear p-mTOR
n (%)
p-value
n (%)
p-value
n (%)
p-value
n (%)
48 (48%) 39 (44%) 1 (50%)
N/S
40 (40%) 31 (35%) 1 (50%)
N/S
21 (21%) 20 (23%) 0
N/S
24 (24%) 17 (19%) 1 (50%)
55 (52%) 32 (42%) 1 (20%)
N/S
41 (39%) 28 (36%) 3 (60%)
N/S
24 (23%) 13 (17%) 4 (80%)
N/S
22 (21%) 19 (25%) 1 (20%)
32 (40%) 56 (55%)
0.009
33 (41%) 39 (38%)
N/S
31 (39%) 10 (10%)
<0.001
16 (20%) 26 (25%)
41 (51%) 46 (44%) 1 (25%)
N/S
32 (40%) 38 (37%) 2 (50%)
N/S
17 (21%) 23 (22%) 1 (25%)
N/S
18 (22%) 22 (21%) 2 (50%)
73 (48%) 10 (34%) 5 (71%) 0
N/S
62 (41%) 8 (28%) 2 (29%) 0
N/S
28 (18%) 11 (38%) 2 (29%) 0
N/S
37 (24%) 5 (17%) 0 0
28 (48%) 60 (46%)
N/S
22 (38%) 50 (38%)
N/S
16 (28%) 25 (19%)
N/S
12 (21%) 30 (23%)
p-value N/S
N/S
N/S
N/S
N/S
N/S
may possibly be attributed to the lack of reproducibility in determining this tumor characteristic. A potentially important finding in this study was that the putative active form of mTOR, i.e., p-mTOR, was differentially expressed in triple negative and non-triple negative cancers. In particular, nuclear p-mTOR was expressed more frequently in the triple-negative than non-triple negative samples. In a previous study using ovarian granulosa cells in culture, Yaba et al14 reported that p-mTOR correlated with the active status of mTOR. Using breast cancer cell lines in culture, Vazquez-Martin found that that the nuclear accumulation of a specific form of p-mTOR closely correlated with proliferative capacity.15 Furthermore, mTOR was mostly cytoplasmic and perinuclear in non-dividing cells but was tightly localised to the mitotic spindle in M-phase cells. These studies therefore suggest that the presence of p-mTOR corresponds with the activation of mTor and an increase in proliferation. In a further study, Tsang et al16 found that nuclear localized mTOR mediated transcription of rDNA and tRNA, nucleic acids that are critical for protein synthesis. These findings when taken together suggest that nuclear mTOR is the main biological form of this protein. Our results thus suggest that active nuclear mTOR plays a more important role in the development and/or progression of triple-negative than non-triple negative breast cancers. Consequently, triple-negative tumours may be more sensitive than non triple-negative tumors to treatment with inhibitors that target mTOR. In conclusion, our finding of high expression of nuclear p-mTOR in approximately 36% of triple-negative breast carcinomas suggests
Table 3 Comparative expression p-mTOR in triple negative (TN) and non-TN breast cancers. NS, not significant; PN, perinuclear. Status
No. of patients
Positive for nuclear p-mTOR n (%)
Positive for PN p-mTOR n (%)
Positive for cytoplasmic p-mTOR n (%)
TN Non TN p-value
88 99
32 (36) 10 (10) <0.001
4 (4) 37 (37) <0.001
33 (37.5) 39 (39) NS
that inhibitors directed against this protein may be effective in at least some patients with this subtype of breast cancer. Patients likely to respond may be those expressing high levels of mTOR/pmTOR. This hypothesis should now be considered for testing in clinical trials. Conflict of interest statement The authors have no conflicts of interest to declare. Ethical approval Ethical approval for this study was obtained from the St, Vincent’s University Hospital Ethics Committee. Acknowledgement The authors wish to thank the Science Foundation Ireland, Strategic Research Cluster award (08/SRC/B1410) to Molecular Therapeutics for Cancer Ireland for funding this work. The sponsors had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript or the decision to submit the manuscript for publication. References 1. Podo F, Buydens LMC, Degani H, Buydens LM, Degani H, Hilhorst R, et al. Triple-negative breast cancer: present challenges and new perspectives. Mol Oncol. 4(3): p. 209e229. 2. Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N. Triple-negative breast cancerecurrent status and future directions. Ann Oncol 2009;20(12):1913e27. 3. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006;441(7092):424e30. 4. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci 2009;122(Pt 20):3589e94. 5. Courtney KD, Corcoran RB, Engelman JA. The PI3K pathway as drug target in human cancer. J Clin Oncol. 28(6): pp. 1075e1083. 6. Alvarez RH, Valero V, Hortobagyi GN. Emerging targeted therapies for breast cancer. J Clin Oncol. 28(20): pp. 3366e3379. 7. Mabuchi S, Kawase C, Altomare DA, Morishige K, Sawada K, Hayashi M, et al. mTOR is a promising therapeutic target both in cisplatin-sensitive and
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