Breast Cancer Res Treat (2015) 150:91–103 DOI 10.1007/s10549-015-3308-4
PRECLINICAL STUDY
The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways Dena A. Jerjees • Ola H. Negm • M. Layth Alabdullah • Sameer Mirza • Methaq Alkaabi • Mohamed R. Hameed • Rezvan Abduljabbar • Abir Muftah Chris C. Nolan • Andrew R. Green • Patrick J. Tighe • Vimla Band • Ian O. Ellis • Emad A. Rakha
•
Received: 6 February 2015 / Accepted: 9 February 2015 / Published online: 21 February 2015 Ó Springer Science+Business Media New York 2015
Abstract The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER? and ER- cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic
of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER?HER2subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression. Keywords mTORC1 Breast cancer pi3k pathway Immunohistochemistry
Introduction Breast cancer (BC) is a major disease that needs deciphering of its different clinical and molecular aspects to fully understand its predisposing factors, molecular background and
Ola H Negm: First joint author. D. A. Jerjees (&) M. Alkaabi R. Abduljabbar A. Muftah C. C. Nolan A. R. Green I. O. Ellis E. A. Rakha Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK e-mail: [email protected] D. A. Jerjees Department of Pathology, Mosul School of Medicine, University of Mosul, Mosul, Iraq
O. H. Negm M. R. Hameed Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt M. L. Alabdullah Academic Unit of Clinical Oncology, School of Medicine, Nottingham City Hospital, The University of Nottingham, Nottingham, UK S. Mirza V. Band Department of Genetics, Cell Biology and Anatomy, University of Nebraska, Lincoln, USA
O. H. Negm M. R. Hameed P. J. Tighe School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, UK
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prognosis. As phenotypically similar tumours behave differently, novel markers and techniques need to contribute to the possible early detection and categorisation of BC [1]. Oestrogen receptor (ER) is a key factor in predicting the response of hormonal therapy in BC [2]. Nevertheless, not all patients respond similarly and up to 50 % of ER? patients show resistant after treatment [3, 4]. The detection of certain proteins that have an association with ER and even HER2 is therefore important. The activated mammalian target of rapamycin complex 1 (mTORC1) is one target that has a distinct role in association with key markers of BC and plays a role in controlling cell division, motility and survival. For instance, PI3K-AKTmTOR pathway can have an impact on influencing the behaviour of different cells even though this pathway has different interactions with other key pathways including ER, HER2 [5, 6] and mitogen-activated protein kinase pathway (MAPK). Such interactions are thought to be responsible for the different behaviours encountered and may influence certain interactions that lead to prognostic differences. For example, using MEK inhibitors to target MAPK pathway in BC inhibits members of this pathway like ERK1/2 but will stimulate PI3 K/Akt/mTOR pathway [7]. mTOR inhibitors that target the PI3K/Akt/mTOR pathway are the first of the targeted therapies to be evaluated in clinical trials to overcome endocrine resistance. Furthermore, another possibility of resistance to treatment is the availability of positive feedback stimulation of the upstream mediators when lower effectors are inhibited. For instance by inhibiting AKT and mTORC1, there is stimulation of HER1 and HER3 proteins in trastuzumabsensitive and -resistant cell lines [7, 8]. The latter study gives an indication of combined therapy rather than using a single one [9]. This interplay indicates that there are certain networks between different BC pathways that are dynamically linking them and that perturbations in one pathway may have consequences on others and eventually on the efficacy of targeted therapy [7]. Several factors can stimulate mTORC1 including growth factors which can enhance the upstream effector PI3K either directly or indirectly through certain docking proteins like insulin receptor substrate or GRB2-associated binder. Other factors that can stimulate p-mTORC1 include a status of energy, amino acids levels and cellular stress [10, 11]. PI3K stimulates AKT and downstream effectors in promoting cell survival proliferation and motility [10]. In mammalian tissues, two proteins have been identified: mTORC1 and mTORC2, both containing atypical serine/ threonine kinase, which belong to the PI3K-like kinase family, which influences a variety of cellular functions. Such kinase is called mTOR/FRAP1 but with RAPTOR (a scaffolding protein) it forms the mTORC1 and with RICTOR (mTORC2-associated scaffolding protein) forms
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mTORC2, in addition also to another mammalian stressactivated protein kinase-interacting protein [11–13]. Furthermore, p-mTORC1 is responsible for the phosphorylation of two important regulators of protein translation: p70S6K (ribosomal p70S6 kinase) and 4E-BP (eukaryotic initiation factor 4E-binding protein) [12, 14]. Another study has shown that oestrogen can enhance the activation of mTOR pathway in oestrogen target tissues such as BC [15]. However, this activation is only demonstrated either by regulation of upstream or downstream mediators of mTOR rather than phosphorylation of the latter [16]. Meanwhile, the PI3K/Akt/mTOR pathway lies downstream to HER2 and aberrantly this pathway can be activated either due to a mutation of the PI3KCA gene or through loss of phosphatase and tensin homolog (PTEN) expression, both seemingly to affect anti-HER2 therapy regimens [5, 6]. Meanwhile, the role of intact ER pathway in upregulating p-mTORC1 using MCF-7 cell line has been shown. Inversely, p-mTORC1 stimulates ER by up to 6-fold [17]. In addition, although some studies showed that p-mTORC1 is associated with worse prognosis and could be a target for BC therapy, other studies revealed different results [17]. It is important to mention that although different studies implicate the usefulness of mTORC1 inhibitors in BC management, efforts are limited in identifying the most useful biomarkers that can guide which patients can benefit the most from this proposed therapy. Interestingly, the TRAMAD trial, which is a translational study, has identified everolimus to be a useful mTORC1 inhibitor in those with low PI3K [18]. Moreover, a biomarker retrospective study of the BOLERO-2 trial has used next-generation sequencing to identify 4 common pathways which are linked with response to everolimus treatment. Furthermore, everolimus is more useful in BC patients with minimal genetic changes of PI3K [19]. The aim is to investigate the protein expression of p-mTORC1 in a large BC cohort and to correlate its expression with clinicopathological variables and proteins related to ER and HER2 pathways. In addition, it is aimed to measure the expression of the protein mTORC1 together with PI3K and Akt in BC cell lines, with differential expression of ER and HER2, using reverse phase protein array (RPPA) technique and to compare the results obtained with immunohistochemistry (IHC). RPPA is an evolving technique and could be used to be an alternative for gene expression profiling as it reflects the proteasome [20]. This retrospective study adheres to REMARK criteria [21].
Materials and methods In this study, 1300 cases were available for the assessment of the expression of p-mTORC1 in a BC cohort prepared as
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tissue microarray (TMA). This cohort is the Nottingham Tenovus primary invasive BC series which is an unselected and consecutive series of patients enrolled between 1988 and 1998. All patients had primary operable disease at presentation and were\70 years of age with a mean age of 55 years; moreover, all had their lymph node stage between I and III. Clinicopathological information including patients’ age, menopausal status, tumour characteristics in form of grade, stage, type, size and Nottingham prognostic index (NPI) was collected [22]. The treatment in this series was to consider NPI score and ER status. If the patient was ER-positive and the NPI was \3.4, no adjuvant hormonal therapy was recommended. While, in ER-positive patients who had NPI score of[3.4, adjuvant hormonal therapy was considered in form of tamoxifen (?Zoladex if the patient is premenopausal). Finally, the systemic chemotherapy in the form of Cyclophosphamide, Methotrexate and 5-Flurouracil (CMF) was given for those with ER-negative tumours if they were fit enough for this treatment regimen. Therefore, the management strategy was uniform for all the patients. Moreover, none of the patients received neoadjuvant therapy or anti-HER2 treatments. Another characteristic of this series is the prospective collection of the outcome data. These include the breast cancer-specific survival (BCSS) which is the time in months from the BC surgery until the BC-associated death occurs (mean = 123 months), while distant metastasis-free survival (DMFS) is the time in months from the date of surgery until the development of distant metastasis (mean = 113). Data regarding the relevant different biomarkers used in this study were also available [23–27]. Immunohistochemistry Four lm sections were cut onto Xtra Slides (Surgipath) from the TMA blocks, then heated to 60 °C in order to enhance adherence. Deparaffinisation in xylene was done twice for 5 min, rehydrated in 100 % alcohol for 2 min 9 3 and rinsed them in tap water for 5 min. Microwave antigen retrieval for 20 min was used for heating slides in 0.01 M citrate buffer (pH6). Manual immunohistochemical staining was performed using Novocastra Novolink Polymer Detection System (Leica Microsystems, Newcastle, UK). Blocking peroxidase activity (endogenous) in the sections was performed using hydrogen peroxide for 5 min. Then optimised primary antibodies including p-mTORC1 [primary rabbit monoclonal antibody (Ser2248), clone (49F9), Cell Signalling] was incubated overnight at 4 °C; primary rabbit anti-PIK3CA antibody (Prestige antibodies powered by Atlas, product No. HPA009985, Sigma, Aldrich, UK) and rabbit polyclonal anti-phospho Akt antibody (Ser473, Cell Signalling) were incubated for 60 min at room temperature.
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Polymer link was applied for 30 min followed by enzyme substrate for 5 min. Haematoxylin was used for counterstaining purposes for 5 min and was followed by rinsing with tap water, dehydration in alcohol, clearing in xylene and mounting with cover slips using DPX (BDH, Poole, UK). Details of other antibodies used in the analysis for comparison are in previous publications [23, 24, 28]. Scoring of immunohistochemistry Visual scoring of TMA slides was performed using highresolution digital images (NanoZoomer, Hamamatsu Photonics Welwyn Garden City, UK) obtained at 2009 magnification, using a web-based interface (Distiller, Slidepath Ltd., Dublin, Ireland). The H-score method was used to score the antibody as it is the representative for the intensity and the percentage. The X-tile software [29] was used to determine categorical cut-points and for p-mTORC1 it was chosen as [30 H-score. Source of cell lines used in this study Wild-type MCF-7 (ER?HER2-), MDA-MB-231 (ERHER2-), SKBR3 (ER-HER2?) and BT474 (ER?HER2?) were provided by the American Type Culture Collection (Manassas, VA). MCF-7, BT474 and MDA-231 cells were cultured in RPMI media, while SKBR3 in McCoy’s media and all of them were provided with 10 % foetal bovine serum. Retroviral infections In order to investigate the effect of HER2 on MAPKs, wild-type HER2-negative cell lines (MCF-7 and MDAMB-231) were transfected with wild-type ErbB2 construct [30] to generate ErbB2 overexpressing cell lines. Retroviral supernatants were generated by calcium phosphate-mediated cotransfection of the expression plasmids and the packaging plasmid pIK into the packaging cell line TSA54 [30]. The retroviral supernatants, collected 24 h after transfection, were used to infect subconfluent MCF7 and MDA-MB-231 in three sequential 4-hours incubations in the presence of 4 lg/mL polybrene (Sigma-Aldrich). Transductants were selected in puromycin 0.5 lg/mL, 48 h after infection. The generated transductants were always cultured in the selection media [30]. Transfected cell lines, MCF-7-HER2? (transfected ER?HER2?) and MDA-MB-231-HER2? (transfected ER-HER2?), were maintained in MEMa media (RPMI supplemented with 10 % FBS, 1 % MEM sodium pyruvate (GIBCO, BRL), 1 % HEPES (GIBCO, BRL) and 1 % Lglutamine (GIBCO, BRL) and antibiotics). All cell lines were grown in a humidified incubator in the presence of 5 % CO2 at 37 °C.
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Growing of the cells and preparation of cell lysate The same method was used as previously published [31]. RPPA technique RPPA methodology has been established before [32–34]. In brief, cell lysates were solubilised in 49 SDS r, using a ratio of 3:1, respectively, and heating them for 5 min at 95 °C. Samples were loaded onto a 384-well plate (Genetix, UK), where each sample was serially diluted 5 times in 19 SDS buffer. Samples were robotically spotted, in duplicate, onto nitrocellulose-coated glass slides (Grace Bio-Labs, USA) using a microarrayer (MicroGrid 610, Digilab, Marlborough, MA, USA). Slides were blocked with I-Block solution (0.2 % I-block (Tropix, Bedford, MA, USA) and 0.1 % Tween-20 in PBS) overnight at 4 °C with shaking. After washing with Tris buffer saline tween (TBST) three times for 5 min each, the slides were incubated with anti-p-mTORC1 (Cell Signalling Technology) and diluted 1:250 in antibody diluent (DAKO). In addition, mouse b-actin (Sigma-Aldrich, UK) diluted 1:1000 was used as a housekeeping protein to control protein loading. Slides were incubated overnight at 4 °C with shaking. Following washing, the slides were incubated for 30 min at room temperature with infrared
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(1:5000 in washing buffer) secondary antibodies (800CW anti-rabbit and 700CW anti-mouse antibodies) from LICOR, Biosciences. After washing and drying, the slides were scanned with a Licor Odyssey scanner at 21 lm resolution at 800 nm (green) and 700 nm (red). The resultant TIFF images were processed with Axon Genepix Pro-6 Microarray Image Analysis software (Molecular Services Inc.) in order to obtain fluorescence data for each feature and to generate gpr files. The signals of protein were determined, after subtraction of the background and normalisation to the internal housekeeping target, using RPPanalyzer, a module within the statistical language on the CRAN (http://cran.rproject.org/) [35]. Finally, using Multi Experiment Viewer (MEV) software, the heat maps were created. Statistical analysis SPSS v21 statistical software (SPSS Inc., Chicago, IL, USA) was used for performing the analysis. Chi squared test was used for finding the associations between different categorical markers. RPPA data were analysed using oneway ANOVA test. Survival curves of BCSS and DMFS were performed using Kaplan–Meier and the significance was estimated using Log-Rank tests. A two-tailed P value of \0.05 was considered to be significant.
Fig. 1 p-mTORC1 expression in BC tissue, A negative, B weak, C moderate and D strong immunohistochemical staining
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Table 1 The association between p-mTOR and the clinicopathological variables in the whole series and ER?HER2- tumours Whole series Low N (%)
ER?HER2- tumours High N (%)
P value
NS
Low N (%)
High N (%)
P value
0.023
Age \50
242 (36)
186 (34)
[50
435 (64)
359 (66)
Pre-
271 (40)
209 (38)
Post-
414 (60)
338 (62)
310 (46)
286 (53)
363 (54)
254 (47)
1
420 (62)
323 (60)
2
197 (29)
183 (34)
3
58 (9)
35 (6)
1
89 (13)
106 (20)
81 (21)
94 (22)
2
189 (28)
232 (43)
150 (40)
212 (51)
3
394 (59)
202 (37)
149 (39)
112 (27)
1
27 (4)
35 (7)
24 (6)
32 (8)
2
191 (29)
211 (40)
143 (39)
176 (44)
3
434 (67)
277 (53)
201 (55)
196 (48)
14 (2)
11 (2)
13 (4)
10 (3)
2
203(31)
248 (48)
174 (47)
224 (55)
3
435 (67)
263 (50)
181 (49)
170 (42)
1
184 (28)
225 (43)
156 (42)
210 (52)
2
120 (18)
114 (22)
89 (24)
92 (23)
3
348 (53)
184 (35)
123 (34)
102 (25)
Probable/Negative
432 (65)
365 (68)
249 (65)
288 (69)
Definite
238 (35)
171 (32)
133 (35)
128 (31)
169 (26)
201 (39)
142 (39)
179 (45)
Moderate prognostic group
365 (57)
253 (49)
181 (50)
181 (45)
Poor prognostic group
112 (17)
66 (12)
39 (11)
42 (10)
99 (26)
140 (33)
284 (74)
282 (67)
120 (31)
153 (36)
262 (69)
268 (64)
198 (52)
231 (55)
184 (48)
188 (45)
246 (65)
247 (59)
110 (29)
146 (35)
25 (6)
25 (6)
Menopausal Status
Tumour Size (cm) \2.0 C2.0
NS
0.017
NS
NS
Stage NS
NS
Grade 1 9 1027
0.001
Tubules
Pleomorphism 1
1 9 1025
1 9 1027
NS
0.069
Mitosis 1 9 1027
0.016
Lymphovascular invasion NS
NS
NPI Good prognostic group
1 9 1027
0.319
Bold P values denote significant one, 0.050–0.09 are borderline associations and NS is [090
Results In this study, 1236 cases were informative for p-mTORC1 immunohistochemical assessment. The expression was cytoplasmic with no nuclear or membranous staining observed. The staining was heterogeneous and the intensity ranged from negative, weak, moderate and intense (Fig. 1). The staining was observed in normal breast epithelium and in DCIS present in TMA cores. 55.7 % cases showed
negative/low expression and 44.3 % had moderate/high expression. Association between p-mTORC1 and clinicopathological variables p-mTORC1 was positively associated with clinicopathological parameters and characteristics of good prognosis including smaller tumour size (P = 0.017), lower tumour
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Table 2 The association between p-mTORC1 and different proteins related to ER and HER2 pathways Whole series Low, N (%)
ER?HER2- tumours High, N (%)
P value
Low, N (%)
High, N (%)
P value
Hormone Receptors and ER-related proteins ER
–
Negative
218 (32)
70 (13)
Positive
458 (68)
468 (87)
311 (48)
157 (30)
340 (52)
363 (70)
Negative
257 (43)
132 (28)
Positive
346 (57)
337 (72)
–
–
1 9 1027
PgR Negative Positive AR
1 9 1027
1 9 1026
80 (21)
79 (19)
300 (79)
337 (81)
86 (24)
85 (22)
270 (76)
294 (78)
NS
1 9 1026
CK7/8 Negative
16 (3)
1 (0)
Positive
630 (97)
520 (100)
Negative
115 (19)
21 (5)
Positive
480 (81)
440 (95)
Negative
86 (14)
28 (6)
Positive
549 (86)
482 (94)
0.001
1 (0)
0 (0)
380 (100)
413 (100)
NS
CK18 1 9 1027
23 (7)
8 (2)
328 (93)
362 (98)
0.004
CK19 6 9 1026
23 (6)
17 (4)
351 (94)
387 (96)
112 (48)
117 (45)
122 (52)
145 (55)
83 (34)
66 (25)
NS
FOXA1 Negative
254 (60)
163 (48)
170 (40)
174 (52)
Negative
157 (38)
80 (24)
Positive
258 (62)
248 (76)
Negative
208 (56)
125 (42)
Positive
166 (44)
172 (58)
Negative
207 (54)
123 (41)
Positive
176 (46)
180 (59)
Negative
272 (72)
156 (52)
Positive
108 (28)
142 (48)
Negative
193 (43)
156 (46)
Positive
257 (57)
185 (54)
CARM1 Negative
113 (27)
85 (25)
80 (34)
72 (28)
Moderate
195 (47)
180 (54)
116 (48)
136 (53)
High
105 (26)
71 (21)
43 (18)
50 (19)
Positive BEX1
0.001
9 9 1025
161 (66.0 %)
195 (75)
109 (51)
101 (44)
103 (49)
129 (56)
NS
0.032
TFF1 4 9 1024
NS
TFF3 4 9 1024
97 (43)
97 (41)
127 (57)
141 (59)
117 (56)
102 (44)
91 (44)
129 (56)
NS
GATA3 1 9 1027
1 9 1027
CD71 NS
NS
133 (53)
136 (52)
118 (47)
128 (48)
0.011
NS
PELP1 Negative
71 (16)
61 (18)
53 (22)
51 (19)
Moderate
291 (66)
224 (65)
165 (64)
182 (67)
82 (18)
60 (17)
38 (14)
38 (14)
High
123
NS
NS
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Table 2 continued Whole series Low, N (%)
ER?HER2- tumours High, N (%)
P value
Low, N (%)
High, N (%)
P value
Proteins of epithelial mesenchymal transition (EMT), tumour suppressor, proliferation, apoptosis and HER family proteins E-Cadherin Negative
237 (37)
178 (35)
Positive
397 (63)
329 (65)
234 (43)
230 (56)
316 (57)
183 (44)
435 (68)
394 (77)
208 (32)
115 (23)
NS
129 (35)
145 (36)
244 (65)
260 (64)
197 (62)
212 (64)
123 (38)
122 (36)
304 (80)
343 (84)
75 (20)
66 (16)
132 (42)
117 (34)
179 (58)
226 (66)
162 (51)
192 (56)
153 (49)
152 (44)
NS
p-Cadherin Negative Positive p53 Negative/low Positive
5 9 1025
2 9 1024
5 9 1025
2 9 1024
BRCA1 Negative/low
269 (50)
167 (40)
High
270 (50)
252 (60)
Negative/low
195 (37)
217 (49)
High
335 (63)
224 (51)
Negative/low
213 (45)
129 (34)
High
262 (55)
251 (66)
0.002
0.028
KI67-LI 9 9 1025
NS
BCL2 0.001
66 (24)
77 (26)
213 (76)
223 (74)
NS
p-AKT Negative/low
131 (29)
65 (19)
316 (71)
278 (81)
101 (20)
88 (22)
Moderate
136 (27)
136 (35)
98 (33)
120 (39)
High
268 (53)
166 (42)
134 (44)
109 (36)
Negative
482 (75)
449 (86)
314 (84)
370 (90)
Positive
161 (25)
72 (14)
60 (16)
40 (10)
Negative
548 (86)
462 (89)
Positive
92 (14)
57 (11)
Negative
47 (8)
37 (8)
Positive
551 (92)
430 (92)
High p-PI3K Negative/low
0.001
62 (24)
51 (19)
193 (76)
223 (81)
69 (23)
78 (25)
NS 0.071
0.006
HER1 2 9 1026
HER2
–
–
0.008 –
0.086
HER3 NS
58 (156)
57 (14)
314 (84)
352 (86)
314 (84)
370 (90)
60 (16)
40 (10)
NS
HER4 Negative
80 (13)
62 (12)
Positive
557 (87)
452 (88)
NS
grade (P = 1 9 1027) with more tubule formation (P = 1 9 1025), less pleomorphism, low mitotic count (P = 1 9 1027 and P = 1 9 1027) and lower NPI score (P = 1 9 1027) but not with lymph node status. These associations were observed in the whole series (Table 1) and in luminal (ER?HER2-) class (Table 1) but not in the HER2-positive (HER2?ER- and HER2?ER?) cohorts.
0.008
Association between p-mTORC1 and key BC biomarkers p-mTORC1 was positively associated with clinicopathological variables, (Table 1) ER and related proteins but negatively with HER2 (Table 2). However, when ER expression is lost, p-mTORC1 showed positive association with HER2, (Table 3).
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Table 3 The association between HER2 and proteins of PI3K/Akt pathways in ER? and ER- BC
Table 4 The association between p-mTORC1 and different proteins related to ER and HER2 pathways within ER-HER2? disease
Biological markers
Biological markers
HER2 Negative N (%)
Positive N (%)
P value
Within ER? BC 625 (91) 62 (9)
Negative/low
235 (25)
16 (17)
329 (35)
26 (27)
High
367 (40)
54 (56)
p-mTORC1
0.279
0.006
383 (88)
422 (94)
52 (12)
29 (6)
12 (40) 18 (60)
3 (15) 17 (85)
0.052
0.023
Negative
19 (76)
7 (41)
Positive
6 (24)
10 (59)
BRCA1 Negative/low
18 (55)
17 (85)
High
15 (45)
3 (15)
0.023
Proliferation markers: KI67-LI
p-AKT
Negative/low
Negative/low
86 (88)
135 (68)
High
12 (12)
63 (32)
2 9 1024
4 (14)
8 (33)
25 (86)
16 (66)
Negative
28 (72)
14 (50)
Positive
11 (28)
14 (50)
High
0.090
HER family proteins HER1
p-PI3K Moderate High
Negative Positive
Tumour suppressor proteins 0.004
Within ER- BC
Negative/low
P value
TFF1
Moderate
High
High N (%)
BEX1 167 (88) 22 (12)
p-PI3K
Negative/low
Low N (%) Other ER-related proteins
p-AKT Negative/low High
ER-HER2? disease
39 (16)
3 (4)
47 (20) 155 (64)
11 (13) 70 (83)
161 (80)
36 (56)
39 (20)
28 (44)
0.002
0.069
p-mTORC1 Negative/low High
1 9 1024
Within the whole series, high expression of p-mTORC1 was significantly associated with the upregulation of hormone receptors (ER; P = 1 9 1027, progesterone receptor (PgR); P = 1 9 10-7 and androgen receptor (AR); P = 1 9 1026), ER-related proteins including GATA3, FOXA1, TFF1, TFF3 and BEX1 and with the expression of the luminal low-molecular weight cytokeratins CK7/8, CK18 and CK19 (Table 2). A positive association was also observed with Akt, BRCA1 and BCL2 (Table 2). There was an inverse correlation between high expression of p-mTORC1 and proteins associated with poor prognosis including p-cadherin, p53 HER1, PI3K and the proliferation marker ki67. In addition, there was a borderline negative association with HER2 expression (Table 2). Stratifying cases according to ER-positive (?) and ERnegative (-) expression revealed that within ER?, high PI3K expression was positively associated with HER2 amplification, while high p-mTORC1 expression was negatively associated with HER2 amplification (Table 3); moreover, there was a positive association between high p-mTORC1 expression and high p-Akt expression but a borderline inverse association with high PI3K expression (P = 0.023, P = 0.083), respectively. Interestingly, high
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expression of p-Akt, PI3K and p-mTORC1, was positively associated with HER2 amplification within the ER- cohort (Table 3). When the cohort was stratified based on the expression of ER and HER2, high p-mTORC1 expression retained its association with most of the biomarkers within the luminal ER?HER2- class as in the whole series (Table 2) but not in the ER-HER2? class (Table 4) or in ER?HER2? class (data not shown). BC outcome Within the whole series, no significant association between p-mTORC1 expression and outcome in terms of BCSS or DMF was found. However, it is worth noting that mTORC1 showed different associations with outcome in the different classes of BC. For instance, when the BC cohort was stratified based on Akt and PI3K expression, p-mTORC1 was associated with longer survival in the Akt? (Fig. 2) and AKT? PI3K high expression subgroups (Fig. 3). Interestingly, when the cohort was selected to a group who had negative/low mTORC1 expression, Akt was associated with worst survival in terms of BCSS and DMFS, (Fig. 4). When analysis was restricted to the group of patients who received hormone therapy and had lymph node-positive disease, mTORC1 showed an association with shorter BCSS and shorter DMFS (Fig. 5).
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Fig. 2 Breast cancer-specific survival and distant metastasis-free survival for mtorc1 in Akt? cohort
Discussion
Fig. 3 Breast cancer-specific survival for p-mTORC1 in AKT? and in high PI3K expressing cohort
Reverse phase protein array analysis RPPA was used to assess the difference in the expression of p-mTORC1 and its related proteins Akt and PI3K in breast cell lines representing different biological classes. RPPA was consistent with IHC results and revealed that p-mTORC1 expression was higher in ER? cell lines compared with ER- cell lines (Fig. 6). However, there was no difference in the expression of p-mTORC1 after HER2 transfection in either ER? or ER- cells (Fig. 6). RPPA revealed significant difference in the expression of Akt and PI3K based on HER2 status with increased expression in the HER2-positive cell lines (wild and transfected) compared to HER2-negative cell lines, (Fig. 6), respectively.
Despite the development and availability of several inhibitors of mTORC1, there remains a concern regarding the role of PI3K/Akt/mTOR signalling in BC and the adverse effect it could have on outcome. Not surprisingly, many studies emphasised the role of this pathway in causing a resistance in the treatment of hormone-dependent BC [36, 37]. We found that p-mTORC1 was associated with better clinicopathological prognostic factors and is strongly related to ER, ER-related proteins, luminal differentiation markers, Akt and PI3K. Interestingly, these associations were observed within the ER?HER2- luminal cohort. The vast majority of mTORC1-positive tumours were hormone receptor positive, while nearly 50 % of hormone receptor tumours showed mTORC1 positivity. Within ER? tumours, mTORC1 expression showed significant association with ER-related proteins, luminal differentiation markers and other features characteristics of good prognosis. The association between mTORC1 and HER2 was only seen in ER-negative tumours. Our IHC results were also supported by RPPA data which revealed that p-mTORC1 had higher expression in MCF-7 and in SKBR3 in comparison to MCF-7-HER2? and MDA-MB-231, respectively. These findings could reflect the major role that ER could play to maintain the function of p-mTORC1 in the favourable path, the interaction between mTORC1 and ER and how the behaviour of mTORC1 has changed when this ER expression was lost. Many studies of which O’Regan R et al. [38] support the role of intact ER-dependent pathway in maintaining the upregulation of p-mTORC1 in BC in cells depending on oestrogen for their growth and survival, keeping in mind that p-mTORC1 is a major regulator of cell growth and cell
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Fig. 4 BCSS and DMFS for Akt in Negative/low mTORC1 cohort
Fig. 5 BCSS and DMFS for p-mTORC1 in those patients with lymph node-positive disease treated with hormonal therapy
viability. Our data regarding the association between p-mTORC1 and clinicopathological variables and ER and its related proteins were in line with Shrivastav et al. [17], who found that p-mTORC1 was associated with smaller tumour size and better overall survival and recurrence-free survival. Those authors defined the p7-ERa score which is a combination of seven ER-phosphorylated epitopes that can be detected in any one tumour. They confirmed the inverse association between p7-ERa score and p-mTORC1. Since the low p7-ERa score represents more phosphorylation of ERa sites which are associated with good prognosis and outcome, conversely, the high p7-ERa score represents more phosphorylation of ERa sites that are associated with poor prognosis and shorter survival which indicates that p-mTORC1 is associated with good prognosis [17]. Moreover, our results are in line with Beca et al. [39] who found that p-mTORC1 is associated with lower
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Fig. 6 RPPA analysis of p-mTORC1, p-PI3K, and p-Akt in 6 BC cell c lines. MCF-7: ER?HER2-, MCF-7-ERB2: ER?HER2? transfected (T), BT474: ER?HER2? (W), MDA-231: ER-HER2-, MDA-231ERB2: ER-HER2? (T), SKBR3: ER-HER2? (W). Each cell line group is represented by 3 columns and 12 rows, each 2 rows of these represent a replicate in duplicate and each duplicate has been taken in 3 dilutions, only BT474 has 2 duplicates and the first group of MDA231-HER2? represented by only 4 replicates has just been considered for comparison and the next one with its full 6 replicates was considered in our analysis
tumour grade and smaller tumour size. Surprisingly, they found that it is an independent predictor of better overall and DMFS in luminal BC. Consistent with our findings, Martina et al. [40] investigated the impact of p-mTORC1 in regulating the transcription factor EB (TFEB), a member of the bHLH
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leucine-zipper family of transcription factors. This protein is required for the biogenesis of the lysosomes which function to clear damaged organelles and to produce autophagy enzymes when activated [40]. The latter study indicated that p-mTORC1 is a kinase and is located in the lysosomes and when the lysosomes function is intact, p-mTORC1 phosphorylates TFEB by relating it to members of the YWHA (14-3-3) family of proteins and by this, its transcriptional activity for the autophagy enzymes can be maintained within the cytosol. In contrast, TFEB is translocated to the nucleus in case of genetic or pharmacological inhibition of p-mTORC1 as there is a lack of phosphorylation and loss of transcriptional activity. Interestingly, feedback inhibition of the PI3K/AKT pathway can be mediated by S6K1, a downstream of p-mTORC1, when there is chronic insulin stimulation, or loss of the tuberous sclerosis complex (TSC1/2 complex), through the phosphorylation of insulin receptor substrate 1 (IRS1), leading to its degradation [41] [42]. Moreover, a complementary negative feedback loop has been identified in which S6K1 phosphorylates Rictor, leading to decrease AKT activation by p-mTORC2 [43, 44]. These facts support our study by referring to the role of p-mTORC1 in enhancing apoptosis. Regarding outcome, we showed a range of combinations that gave different prognostic results. Firstly, within Akt? cohort, p-mTORC1 was associated with prolonged survival; moreover, when the analysis was within Akt?/ PI3K? cohort, we could obtain a trend association. Within negative/low p-mTORC1 expression, Akt was associated with shorter survival and this was not shown within the whole series but this could imply that p-mTORC1 has some element in improving survival. RPPA was useful in testing the expression of the proteins of interest in this study: p-mTORC1, p-Akt and p-PI3K. It showed a good concordance between our results in IHC and RPPA especially regarding p-Akt and p-PI3K. Even for p-mTORC1, it revealed a difference between ER?HER2-/ER?HER2? and between ER-HER2-/ ER-HER2? although the association was not statistically significant. In conclusion, p-mTORC1 is a downstream signalling molecule that can be differentially upregulated by key markers including ER and HER2 in different molecular classes of BC. Although p-mTORC1 was generally associated with good prognosis, its association with outcome seems to be influenced by other proteins such as Akt and PI3K. Acknowledgments Dena A Jerjees is funded by the higher committee of educational development in Iraq. Conflict of interest
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Ethical standards This study was approved by the Nottingham Research Ethics Committee.
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PRECLINICAL STUDY
The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways Dena A. Jerjees • Ola H. Negm • M. Layth Alabdullah • Sameer Mirza • Methaq Alkaabi • Mohamed R. Hameed • Rezvan Abduljabbar • Abir Muftah Chris C. Nolan • Andrew R. Green • Patrick J. Tighe • Vimla Band • Ian O. Ellis • Emad A. Rakha
•
Received: 6 February 2015 / Accepted: 9 February 2015 / Published online: 21 February 2015 Ó Springer Science+Business Media New York 2015
Abstract The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER? and ER- cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic
of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER?HER2subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression. Keywords mTORC1 Breast cancer pi3k pathway Immunohistochemistry
Introduction Breast cancer (BC) is a major disease that needs deciphering of its different clinical and molecular aspects to fully understand its predisposing factors, molecular background and
Ola H Negm: First joint author. D. A. Jerjees (&) M. Alkaabi R. Abduljabbar A. Muftah C. C. Nolan A. R. Green I. O. Ellis E. A. Rakha Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK e-mail: [email protected] D. A. Jerjees Department of Pathology, Mosul School of Medicine, University of Mosul, Mosul, Iraq
O. H. Negm M. R. Hameed Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt M. L. Alabdullah Academic Unit of Clinical Oncology, School of Medicine, Nottingham City Hospital, The University of Nottingham, Nottingham, UK S. Mirza V. Band Department of Genetics, Cell Biology and Anatomy, University of Nebraska, Lincoln, USA
O. H. Negm M. R. Hameed P. J. Tighe School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, UK
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prognosis. As phenotypically similar tumours behave differently, novel markers and techniques need to contribute to the possible early detection and categorisation of BC [1]. Oestrogen receptor (ER) is a key factor in predicting the response of hormonal therapy in BC [2]. Nevertheless, not all patients respond similarly and up to 50 % of ER? patients show resistant after treatment [3, 4]. The detection of certain proteins that have an association with ER and even HER2 is therefore important. The activated mammalian target of rapamycin complex 1 (mTORC1) is one target that has a distinct role in association with key markers of BC and plays a role in controlling cell division, motility and survival. For instance, PI3K-AKTmTOR pathway can have an impact on influencing the behaviour of different cells even though this pathway has different interactions with other key pathways including ER, HER2 [5, 6] and mitogen-activated protein kinase pathway (MAPK). Such interactions are thought to be responsible for the different behaviours encountered and may influence certain interactions that lead to prognostic differences. For example, using MEK inhibitors to target MAPK pathway in BC inhibits members of this pathway like ERK1/2 but will stimulate PI3 K/Akt/mTOR pathway [7]. mTOR inhibitors that target the PI3K/Akt/mTOR pathway are the first of the targeted therapies to be evaluated in clinical trials to overcome endocrine resistance. Furthermore, another possibility of resistance to treatment is the availability of positive feedback stimulation of the upstream mediators when lower effectors are inhibited. For instance by inhibiting AKT and mTORC1, there is stimulation of HER1 and HER3 proteins in trastuzumabsensitive and -resistant cell lines [7, 8]. The latter study gives an indication of combined therapy rather than using a single one [9]. This interplay indicates that there are certain networks between different BC pathways that are dynamically linking them and that perturbations in one pathway may have consequences on others and eventually on the efficacy of targeted therapy [7]. Several factors can stimulate mTORC1 including growth factors which can enhance the upstream effector PI3K either directly or indirectly through certain docking proteins like insulin receptor substrate or GRB2-associated binder. Other factors that can stimulate p-mTORC1 include a status of energy, amino acids levels and cellular stress [10, 11]. PI3K stimulates AKT and downstream effectors in promoting cell survival proliferation and motility [10]. In mammalian tissues, two proteins have been identified: mTORC1 and mTORC2, both containing atypical serine/ threonine kinase, which belong to the PI3K-like kinase family, which influences a variety of cellular functions. Such kinase is called mTOR/FRAP1 but with RAPTOR (a scaffolding protein) it forms the mTORC1 and with RICTOR (mTORC2-associated scaffolding protein) forms
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mTORC2, in addition also to another mammalian stressactivated protein kinase-interacting protein [11–13]. Furthermore, p-mTORC1 is responsible for the phosphorylation of two important regulators of protein translation: p70S6K (ribosomal p70S6 kinase) and 4E-BP (eukaryotic initiation factor 4E-binding protein) [12, 14]. Another study has shown that oestrogen can enhance the activation of mTOR pathway in oestrogen target tissues such as BC [15]. However, this activation is only demonstrated either by regulation of upstream or downstream mediators of mTOR rather than phosphorylation of the latter [16]. Meanwhile, the PI3K/Akt/mTOR pathway lies downstream to HER2 and aberrantly this pathway can be activated either due to a mutation of the PI3KCA gene or through loss of phosphatase and tensin homolog (PTEN) expression, both seemingly to affect anti-HER2 therapy regimens [5, 6]. Meanwhile, the role of intact ER pathway in upregulating p-mTORC1 using MCF-7 cell line has been shown. Inversely, p-mTORC1 stimulates ER by up to 6-fold [17]. In addition, although some studies showed that p-mTORC1 is associated with worse prognosis and could be a target for BC therapy, other studies revealed different results [17]. It is important to mention that although different studies implicate the usefulness of mTORC1 inhibitors in BC management, efforts are limited in identifying the most useful biomarkers that can guide which patients can benefit the most from this proposed therapy. Interestingly, the TRAMAD trial, which is a translational study, has identified everolimus to be a useful mTORC1 inhibitor in those with low PI3K [18]. Moreover, a biomarker retrospective study of the BOLERO-2 trial has used next-generation sequencing to identify 4 common pathways which are linked with response to everolimus treatment. Furthermore, everolimus is more useful in BC patients with minimal genetic changes of PI3K [19]. The aim is to investigate the protein expression of p-mTORC1 in a large BC cohort and to correlate its expression with clinicopathological variables and proteins related to ER and HER2 pathways. In addition, it is aimed to measure the expression of the protein mTORC1 together with PI3K and Akt in BC cell lines, with differential expression of ER and HER2, using reverse phase protein array (RPPA) technique and to compare the results obtained with immunohistochemistry (IHC). RPPA is an evolving technique and could be used to be an alternative for gene expression profiling as it reflects the proteasome [20]. This retrospective study adheres to REMARK criteria [21].
Materials and methods In this study, 1300 cases were available for the assessment of the expression of p-mTORC1 in a BC cohort prepared as
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tissue microarray (TMA). This cohort is the Nottingham Tenovus primary invasive BC series which is an unselected and consecutive series of patients enrolled between 1988 and 1998. All patients had primary operable disease at presentation and were\70 years of age with a mean age of 55 years; moreover, all had their lymph node stage between I and III. Clinicopathological information including patients’ age, menopausal status, tumour characteristics in form of grade, stage, type, size and Nottingham prognostic index (NPI) was collected [22]. The treatment in this series was to consider NPI score and ER status. If the patient was ER-positive and the NPI was \3.4, no adjuvant hormonal therapy was recommended. While, in ER-positive patients who had NPI score of[3.4, adjuvant hormonal therapy was considered in form of tamoxifen (?Zoladex if the patient is premenopausal). Finally, the systemic chemotherapy in the form of Cyclophosphamide, Methotrexate and 5-Flurouracil (CMF) was given for those with ER-negative tumours if they were fit enough for this treatment regimen. Therefore, the management strategy was uniform for all the patients. Moreover, none of the patients received neoadjuvant therapy or anti-HER2 treatments. Another characteristic of this series is the prospective collection of the outcome data. These include the breast cancer-specific survival (BCSS) which is the time in months from the BC surgery until the BC-associated death occurs (mean = 123 months), while distant metastasis-free survival (DMFS) is the time in months from the date of surgery until the development of distant metastasis (mean = 113). Data regarding the relevant different biomarkers used in this study were also available [23–27]. Immunohistochemistry Four lm sections were cut onto Xtra Slides (Surgipath) from the TMA blocks, then heated to 60 °C in order to enhance adherence. Deparaffinisation in xylene was done twice for 5 min, rehydrated in 100 % alcohol for 2 min 9 3 and rinsed them in tap water for 5 min. Microwave antigen retrieval for 20 min was used for heating slides in 0.01 M citrate buffer (pH6). Manual immunohistochemical staining was performed using Novocastra Novolink Polymer Detection System (Leica Microsystems, Newcastle, UK). Blocking peroxidase activity (endogenous) in the sections was performed using hydrogen peroxide for 5 min. Then optimised primary antibodies including p-mTORC1 [primary rabbit monoclonal antibody (Ser2248), clone (49F9), Cell Signalling] was incubated overnight at 4 °C; primary rabbit anti-PIK3CA antibody (Prestige antibodies powered by Atlas, product No. HPA009985, Sigma, Aldrich, UK) and rabbit polyclonal anti-phospho Akt antibody (Ser473, Cell Signalling) were incubated for 60 min at room temperature.
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Polymer link was applied for 30 min followed by enzyme substrate for 5 min. Haematoxylin was used for counterstaining purposes for 5 min and was followed by rinsing with tap water, dehydration in alcohol, clearing in xylene and mounting with cover slips using DPX (BDH, Poole, UK). Details of other antibodies used in the analysis for comparison are in previous publications [23, 24, 28]. Scoring of immunohistochemistry Visual scoring of TMA slides was performed using highresolution digital images (NanoZoomer, Hamamatsu Photonics Welwyn Garden City, UK) obtained at 2009 magnification, using a web-based interface (Distiller, Slidepath Ltd., Dublin, Ireland). The H-score method was used to score the antibody as it is the representative for the intensity and the percentage. The X-tile software [29] was used to determine categorical cut-points and for p-mTORC1 it was chosen as [30 H-score. Source of cell lines used in this study Wild-type MCF-7 (ER?HER2-), MDA-MB-231 (ERHER2-), SKBR3 (ER-HER2?) and BT474 (ER?HER2?) were provided by the American Type Culture Collection (Manassas, VA). MCF-7, BT474 and MDA-231 cells were cultured in RPMI media, while SKBR3 in McCoy’s media and all of them were provided with 10 % foetal bovine serum. Retroviral infections In order to investigate the effect of HER2 on MAPKs, wild-type HER2-negative cell lines (MCF-7 and MDAMB-231) were transfected with wild-type ErbB2 construct [30] to generate ErbB2 overexpressing cell lines. Retroviral supernatants were generated by calcium phosphate-mediated cotransfection of the expression plasmids and the packaging plasmid pIK into the packaging cell line TSA54 [30]. The retroviral supernatants, collected 24 h after transfection, were used to infect subconfluent MCF7 and MDA-MB-231 in three sequential 4-hours incubations in the presence of 4 lg/mL polybrene (Sigma-Aldrich). Transductants were selected in puromycin 0.5 lg/mL, 48 h after infection. The generated transductants were always cultured in the selection media [30]. Transfected cell lines, MCF-7-HER2? (transfected ER?HER2?) and MDA-MB-231-HER2? (transfected ER-HER2?), were maintained in MEMa media (RPMI supplemented with 10 % FBS, 1 % MEM sodium pyruvate (GIBCO, BRL), 1 % HEPES (GIBCO, BRL) and 1 % Lglutamine (GIBCO, BRL) and antibiotics). All cell lines were grown in a humidified incubator in the presence of 5 % CO2 at 37 °C.
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Growing of the cells and preparation of cell lysate The same method was used as previously published [31]. RPPA technique RPPA methodology has been established before [32–34]. In brief, cell lysates were solubilised in 49 SDS r, using a ratio of 3:1, respectively, and heating them for 5 min at 95 °C. Samples were loaded onto a 384-well plate (Genetix, UK), where each sample was serially diluted 5 times in 19 SDS buffer. Samples were robotically spotted, in duplicate, onto nitrocellulose-coated glass slides (Grace Bio-Labs, USA) using a microarrayer (MicroGrid 610, Digilab, Marlborough, MA, USA). Slides were blocked with I-Block solution (0.2 % I-block (Tropix, Bedford, MA, USA) and 0.1 % Tween-20 in PBS) overnight at 4 °C with shaking. After washing with Tris buffer saline tween (TBST) three times for 5 min each, the slides were incubated with anti-p-mTORC1 (Cell Signalling Technology) and diluted 1:250 in antibody diluent (DAKO). In addition, mouse b-actin (Sigma-Aldrich, UK) diluted 1:1000 was used as a housekeeping protein to control protein loading. Slides were incubated overnight at 4 °C with shaking. Following washing, the slides were incubated for 30 min at room temperature with infrared
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(1:5000 in washing buffer) secondary antibodies (800CW anti-rabbit and 700CW anti-mouse antibodies) from LICOR, Biosciences. After washing and drying, the slides were scanned with a Licor Odyssey scanner at 21 lm resolution at 800 nm (green) and 700 nm (red). The resultant TIFF images were processed with Axon Genepix Pro-6 Microarray Image Analysis software (Molecular Services Inc.) in order to obtain fluorescence data for each feature and to generate gpr files. The signals of protein were determined, after subtraction of the background and normalisation to the internal housekeeping target, using RPPanalyzer, a module within the statistical language on the CRAN (http://cran.rproject.org/) [35]. Finally, using Multi Experiment Viewer (MEV) software, the heat maps were created. Statistical analysis SPSS v21 statistical software (SPSS Inc., Chicago, IL, USA) was used for performing the analysis. Chi squared test was used for finding the associations between different categorical markers. RPPA data were analysed using oneway ANOVA test. Survival curves of BCSS and DMFS were performed using Kaplan–Meier and the significance was estimated using Log-Rank tests. A two-tailed P value of \0.05 was considered to be significant.
Fig. 1 p-mTORC1 expression in BC tissue, A negative, B weak, C moderate and D strong immunohistochemical staining
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Table 1 The association between p-mTOR and the clinicopathological variables in the whole series and ER?HER2- tumours Whole series Low N (%)
ER?HER2- tumours High N (%)
P value
NS
Low N (%)
High N (%)
P value
0.023
Age \50
242 (36)
186 (34)
[50
435 (64)
359 (66)
Pre-
271 (40)
209 (38)
Post-
414 (60)
338 (62)
310 (46)
286 (53)
363 (54)
254 (47)
1
420 (62)
323 (60)
2
197 (29)
183 (34)
3
58 (9)
35 (6)
1
89 (13)
106 (20)
81 (21)
94 (22)
2
189 (28)
232 (43)
150 (40)
212 (51)
3
394 (59)
202 (37)
149 (39)
112 (27)
1
27 (4)
35 (7)
24 (6)
32 (8)
2
191 (29)
211 (40)
143 (39)
176 (44)
3
434 (67)
277 (53)
201 (55)
196 (48)
14 (2)
11 (2)
13 (4)
10 (3)
2
203(31)
248 (48)
174 (47)
224 (55)
3
435 (67)
263 (50)
181 (49)
170 (42)
1
184 (28)
225 (43)
156 (42)
210 (52)
2
120 (18)
114 (22)
89 (24)
92 (23)
3
348 (53)
184 (35)
123 (34)
102 (25)
Probable/Negative
432 (65)
365 (68)
249 (65)
288 (69)
Definite
238 (35)
171 (32)
133 (35)
128 (31)
169 (26)
201 (39)
142 (39)
179 (45)
Moderate prognostic group
365 (57)
253 (49)
181 (50)
181 (45)
Poor prognostic group
112 (17)
66 (12)
39 (11)
42 (10)
99 (26)
140 (33)
284 (74)
282 (67)
120 (31)
153 (36)
262 (69)
268 (64)
198 (52)
231 (55)
184 (48)
188 (45)
246 (65)
247 (59)
110 (29)
146 (35)
25 (6)
25 (6)
Menopausal Status
Tumour Size (cm) \2.0 C2.0
NS
0.017
NS
NS
Stage NS
NS
Grade 1 9 1027
0.001
Tubules
Pleomorphism 1
1 9 1025
1 9 1027
NS
0.069
Mitosis 1 9 1027
0.016
Lymphovascular invasion NS
NS
NPI Good prognostic group
1 9 1027
0.319
Bold P values denote significant one, 0.050–0.09 are borderline associations and NS is [090
Results In this study, 1236 cases were informative for p-mTORC1 immunohistochemical assessment. The expression was cytoplasmic with no nuclear or membranous staining observed. The staining was heterogeneous and the intensity ranged from negative, weak, moderate and intense (Fig. 1). The staining was observed in normal breast epithelium and in DCIS present in TMA cores. 55.7 % cases showed
negative/low expression and 44.3 % had moderate/high expression. Association between p-mTORC1 and clinicopathological variables p-mTORC1 was positively associated with clinicopathological parameters and characteristics of good prognosis including smaller tumour size (P = 0.017), lower tumour
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Table 2 The association between p-mTORC1 and different proteins related to ER and HER2 pathways Whole series Low, N (%)
ER?HER2- tumours High, N (%)
P value
Low, N (%)
High, N (%)
P value
Hormone Receptors and ER-related proteins ER
–
Negative
218 (32)
70 (13)
Positive
458 (68)
468 (87)
311 (48)
157 (30)
340 (52)
363 (70)
Negative
257 (43)
132 (28)
Positive
346 (57)
337 (72)
–
–
1 9 1027
PgR Negative Positive AR
1 9 1027
1 9 1026
80 (21)
79 (19)
300 (79)
337 (81)
86 (24)
85 (22)
270 (76)
294 (78)
NS
1 9 1026
CK7/8 Negative
16 (3)
1 (0)
Positive
630 (97)
520 (100)
Negative
115 (19)
21 (5)
Positive
480 (81)
440 (95)
Negative
86 (14)
28 (6)
Positive
549 (86)
482 (94)
0.001
1 (0)
0 (0)
380 (100)
413 (100)
NS
CK18 1 9 1027
23 (7)
8 (2)
328 (93)
362 (98)
0.004
CK19 6 9 1026
23 (6)
17 (4)
351 (94)
387 (96)
112 (48)
117 (45)
122 (52)
145 (55)
83 (34)
66 (25)
NS
FOXA1 Negative
254 (60)
163 (48)
170 (40)
174 (52)
Negative
157 (38)
80 (24)
Positive
258 (62)
248 (76)
Negative
208 (56)
125 (42)
Positive
166 (44)
172 (58)
Negative
207 (54)
123 (41)
Positive
176 (46)
180 (59)
Negative
272 (72)
156 (52)
Positive
108 (28)
142 (48)
Negative
193 (43)
156 (46)
Positive
257 (57)
185 (54)
CARM1 Negative
113 (27)
85 (25)
80 (34)
72 (28)
Moderate
195 (47)
180 (54)
116 (48)
136 (53)
High
105 (26)
71 (21)
43 (18)
50 (19)
Positive BEX1
0.001
9 9 1025
161 (66.0 %)
195 (75)
109 (51)
101 (44)
103 (49)
129 (56)
NS
0.032
TFF1 4 9 1024
NS
TFF3 4 9 1024
97 (43)
97 (41)
127 (57)
141 (59)
117 (56)
102 (44)
91 (44)
129 (56)
NS
GATA3 1 9 1027
1 9 1027
CD71 NS
NS
133 (53)
136 (52)
118 (47)
128 (48)
0.011
NS
PELP1 Negative
71 (16)
61 (18)
53 (22)
51 (19)
Moderate
291 (66)
224 (65)
165 (64)
182 (67)
82 (18)
60 (17)
38 (14)
38 (14)
High
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NS
NS
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Table 2 continued Whole series Low, N (%)
ER?HER2- tumours High, N (%)
P value
Low, N (%)
High, N (%)
P value
Proteins of epithelial mesenchymal transition (EMT), tumour suppressor, proliferation, apoptosis and HER family proteins E-Cadherin Negative
237 (37)
178 (35)
Positive
397 (63)
329 (65)
234 (43)
230 (56)
316 (57)
183 (44)
435 (68)
394 (77)
208 (32)
115 (23)
NS
129 (35)
145 (36)
244 (65)
260 (64)
197 (62)
212 (64)
123 (38)
122 (36)
304 (80)
343 (84)
75 (20)
66 (16)
132 (42)
117 (34)
179 (58)
226 (66)
162 (51)
192 (56)
153 (49)
152 (44)
NS
p-Cadherin Negative Positive p53 Negative/low Positive
5 9 1025
2 9 1024
5 9 1025
2 9 1024
BRCA1 Negative/low
269 (50)
167 (40)
High
270 (50)
252 (60)
Negative/low
195 (37)
217 (49)
High
335 (63)
224 (51)
Negative/low
213 (45)
129 (34)
High
262 (55)
251 (66)
0.002
0.028
KI67-LI 9 9 1025
NS
BCL2 0.001
66 (24)
77 (26)
213 (76)
223 (74)
NS
p-AKT Negative/low
131 (29)
65 (19)
316 (71)
278 (81)
101 (20)
88 (22)
Moderate
136 (27)
136 (35)
98 (33)
120 (39)
High
268 (53)
166 (42)
134 (44)
109 (36)
Negative
482 (75)
449 (86)
314 (84)
370 (90)
Positive
161 (25)
72 (14)
60 (16)
40 (10)
Negative
548 (86)
462 (89)
Positive
92 (14)
57 (11)
Negative
47 (8)
37 (8)
Positive
551 (92)
430 (92)
High p-PI3K Negative/low
0.001
62 (24)
51 (19)
193 (76)
223 (81)
69 (23)
78 (25)
NS 0.071
0.006
HER1 2 9 1026
HER2
–
–
0.008 –
0.086
HER3 NS
58 (156)
57 (14)
314 (84)
352 (86)
314 (84)
370 (90)
60 (16)
40 (10)
NS
HER4 Negative
80 (13)
62 (12)
Positive
557 (87)
452 (88)
NS
grade (P = 1 9 1027) with more tubule formation (P = 1 9 1025), less pleomorphism, low mitotic count (P = 1 9 1027 and P = 1 9 1027) and lower NPI score (P = 1 9 1027) but not with lymph node status. These associations were observed in the whole series (Table 1) and in luminal (ER?HER2-) class (Table 1) but not in the HER2-positive (HER2?ER- and HER2?ER?) cohorts.
0.008
Association between p-mTORC1 and key BC biomarkers p-mTORC1 was positively associated with clinicopathological variables, (Table 1) ER and related proteins but negatively with HER2 (Table 2). However, when ER expression is lost, p-mTORC1 showed positive association with HER2, (Table 3).
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Table 3 The association between HER2 and proteins of PI3K/Akt pathways in ER? and ER- BC
Table 4 The association between p-mTORC1 and different proteins related to ER and HER2 pathways within ER-HER2? disease
Biological markers
Biological markers
HER2 Negative N (%)
Positive N (%)
P value
Within ER? BC 625 (91) 62 (9)
Negative/low
235 (25)
16 (17)
329 (35)
26 (27)
High
367 (40)
54 (56)
p-mTORC1
0.279
0.006
383 (88)
422 (94)
52 (12)
29 (6)
12 (40) 18 (60)
3 (15) 17 (85)
0.052
0.023
Negative
19 (76)
7 (41)
Positive
6 (24)
10 (59)
BRCA1 Negative/low
18 (55)
17 (85)
High
15 (45)
3 (15)
0.023
Proliferation markers: KI67-LI
p-AKT
Negative/low
Negative/low
86 (88)
135 (68)
High
12 (12)
63 (32)
2 9 1024
4 (14)
8 (33)
25 (86)
16 (66)
Negative
28 (72)
14 (50)
Positive
11 (28)
14 (50)
High
0.090
HER family proteins HER1
p-PI3K Moderate High
Negative Positive
Tumour suppressor proteins 0.004
Within ER- BC
Negative/low
P value
TFF1
Moderate
High
High N (%)
BEX1 167 (88) 22 (12)
p-PI3K
Negative/low
Low N (%) Other ER-related proteins
p-AKT Negative/low High
ER-HER2? disease
39 (16)
3 (4)
47 (20) 155 (64)
11 (13) 70 (83)
161 (80)
36 (56)
39 (20)
28 (44)
0.002
0.069
p-mTORC1 Negative/low High
1 9 1024
Within the whole series, high expression of p-mTORC1 was significantly associated with the upregulation of hormone receptors (ER; P = 1 9 1027, progesterone receptor (PgR); P = 1 9 10-7 and androgen receptor (AR); P = 1 9 1026), ER-related proteins including GATA3, FOXA1, TFF1, TFF3 and BEX1 and with the expression of the luminal low-molecular weight cytokeratins CK7/8, CK18 and CK19 (Table 2). A positive association was also observed with Akt, BRCA1 and BCL2 (Table 2). There was an inverse correlation between high expression of p-mTORC1 and proteins associated with poor prognosis including p-cadherin, p53 HER1, PI3K and the proliferation marker ki67. In addition, there was a borderline negative association with HER2 expression (Table 2). Stratifying cases according to ER-positive (?) and ERnegative (-) expression revealed that within ER?, high PI3K expression was positively associated with HER2 amplification, while high p-mTORC1 expression was negatively associated with HER2 amplification (Table 3); moreover, there was a positive association between high p-mTORC1 expression and high p-Akt expression but a borderline inverse association with high PI3K expression (P = 0.023, P = 0.083), respectively. Interestingly, high
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expression of p-Akt, PI3K and p-mTORC1, was positively associated with HER2 amplification within the ER- cohort (Table 3). When the cohort was stratified based on the expression of ER and HER2, high p-mTORC1 expression retained its association with most of the biomarkers within the luminal ER?HER2- class as in the whole series (Table 2) but not in the ER-HER2? class (Table 4) or in ER?HER2? class (data not shown). BC outcome Within the whole series, no significant association between p-mTORC1 expression and outcome in terms of BCSS or DMF was found. However, it is worth noting that mTORC1 showed different associations with outcome in the different classes of BC. For instance, when the BC cohort was stratified based on Akt and PI3K expression, p-mTORC1 was associated with longer survival in the Akt? (Fig. 2) and AKT? PI3K high expression subgroups (Fig. 3). Interestingly, when the cohort was selected to a group who had negative/low mTORC1 expression, Akt was associated with worst survival in terms of BCSS and DMFS, (Fig. 4). When analysis was restricted to the group of patients who received hormone therapy and had lymph node-positive disease, mTORC1 showed an association with shorter BCSS and shorter DMFS (Fig. 5).
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Fig. 2 Breast cancer-specific survival and distant metastasis-free survival for mtorc1 in Akt? cohort
Discussion
Fig. 3 Breast cancer-specific survival for p-mTORC1 in AKT? and in high PI3K expressing cohort
Reverse phase protein array analysis RPPA was used to assess the difference in the expression of p-mTORC1 and its related proteins Akt and PI3K in breast cell lines representing different biological classes. RPPA was consistent with IHC results and revealed that p-mTORC1 expression was higher in ER? cell lines compared with ER- cell lines (Fig. 6). However, there was no difference in the expression of p-mTORC1 after HER2 transfection in either ER? or ER- cells (Fig. 6). RPPA revealed significant difference in the expression of Akt and PI3K based on HER2 status with increased expression in the HER2-positive cell lines (wild and transfected) compared to HER2-negative cell lines, (Fig. 6), respectively.
Despite the development and availability of several inhibitors of mTORC1, there remains a concern regarding the role of PI3K/Akt/mTOR signalling in BC and the adverse effect it could have on outcome. Not surprisingly, many studies emphasised the role of this pathway in causing a resistance in the treatment of hormone-dependent BC [36, 37]. We found that p-mTORC1 was associated with better clinicopathological prognostic factors and is strongly related to ER, ER-related proteins, luminal differentiation markers, Akt and PI3K. Interestingly, these associations were observed within the ER?HER2- luminal cohort. The vast majority of mTORC1-positive tumours were hormone receptor positive, while nearly 50 % of hormone receptor tumours showed mTORC1 positivity. Within ER? tumours, mTORC1 expression showed significant association with ER-related proteins, luminal differentiation markers and other features characteristics of good prognosis. The association between mTORC1 and HER2 was only seen in ER-negative tumours. Our IHC results were also supported by RPPA data which revealed that p-mTORC1 had higher expression in MCF-7 and in SKBR3 in comparison to MCF-7-HER2? and MDA-MB-231, respectively. These findings could reflect the major role that ER could play to maintain the function of p-mTORC1 in the favourable path, the interaction between mTORC1 and ER and how the behaviour of mTORC1 has changed when this ER expression was lost. Many studies of which O’Regan R et al. [38] support the role of intact ER-dependent pathway in maintaining the upregulation of p-mTORC1 in BC in cells depending on oestrogen for their growth and survival, keeping in mind that p-mTORC1 is a major regulator of cell growth and cell
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Fig. 4 BCSS and DMFS for Akt in Negative/low mTORC1 cohort
Fig. 5 BCSS and DMFS for p-mTORC1 in those patients with lymph node-positive disease treated with hormonal therapy
viability. Our data regarding the association between p-mTORC1 and clinicopathological variables and ER and its related proteins were in line with Shrivastav et al. [17], who found that p-mTORC1 was associated with smaller tumour size and better overall survival and recurrence-free survival. Those authors defined the p7-ERa score which is a combination of seven ER-phosphorylated epitopes that can be detected in any one tumour. They confirmed the inverse association between p7-ERa score and p-mTORC1. Since the low p7-ERa score represents more phosphorylation of ERa sites which are associated with good prognosis and outcome, conversely, the high p7-ERa score represents more phosphorylation of ERa sites that are associated with poor prognosis and shorter survival which indicates that p-mTORC1 is associated with good prognosis [17]. Moreover, our results are in line with Beca et al. [39] who found that p-mTORC1 is associated with lower
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Fig. 6 RPPA analysis of p-mTORC1, p-PI3K, and p-Akt in 6 BC cell c lines. MCF-7: ER?HER2-, MCF-7-ERB2: ER?HER2? transfected (T), BT474: ER?HER2? (W), MDA-231: ER-HER2-, MDA-231ERB2: ER-HER2? (T), SKBR3: ER-HER2? (W). Each cell line group is represented by 3 columns and 12 rows, each 2 rows of these represent a replicate in duplicate and each duplicate has been taken in 3 dilutions, only BT474 has 2 duplicates and the first group of MDA231-HER2? represented by only 4 replicates has just been considered for comparison and the next one with its full 6 replicates was considered in our analysis
tumour grade and smaller tumour size. Surprisingly, they found that it is an independent predictor of better overall and DMFS in luminal BC. Consistent with our findings, Martina et al. [40] investigated the impact of p-mTORC1 in regulating the transcription factor EB (TFEB), a member of the bHLH
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leucine-zipper family of transcription factors. This protein is required for the biogenesis of the lysosomes which function to clear damaged organelles and to produce autophagy enzymes when activated [40]. The latter study indicated that p-mTORC1 is a kinase and is located in the lysosomes and when the lysosomes function is intact, p-mTORC1 phosphorylates TFEB by relating it to members of the YWHA (14-3-3) family of proteins and by this, its transcriptional activity for the autophagy enzymes can be maintained within the cytosol. In contrast, TFEB is translocated to the nucleus in case of genetic or pharmacological inhibition of p-mTORC1 as there is a lack of phosphorylation and loss of transcriptional activity. Interestingly, feedback inhibition of the PI3K/AKT pathway can be mediated by S6K1, a downstream of p-mTORC1, when there is chronic insulin stimulation, or loss of the tuberous sclerosis complex (TSC1/2 complex), through the phosphorylation of insulin receptor substrate 1 (IRS1), leading to its degradation [41] [42]. Moreover, a complementary negative feedback loop has been identified in which S6K1 phosphorylates Rictor, leading to decrease AKT activation by p-mTORC2 [43, 44]. These facts support our study by referring to the role of p-mTORC1 in enhancing apoptosis. Regarding outcome, we showed a range of combinations that gave different prognostic results. Firstly, within Akt? cohort, p-mTORC1 was associated with prolonged survival; moreover, when the analysis was within Akt?/ PI3K? cohort, we could obtain a trend association. Within negative/low p-mTORC1 expression, Akt was associated with shorter survival and this was not shown within the whole series but this could imply that p-mTORC1 has some element in improving survival. RPPA was useful in testing the expression of the proteins of interest in this study: p-mTORC1, p-Akt and p-PI3K. It showed a good concordance between our results in IHC and RPPA especially regarding p-Akt and p-PI3K. Even for p-mTORC1, it revealed a difference between ER?HER2-/ER?HER2? and between ER-HER2-/ ER-HER2? although the association was not statistically significant. In conclusion, p-mTORC1 is a downstream signalling molecule that can be differentially upregulated by key markers including ER and HER2 in different molecular classes of BC. Although p-mTORC1 was generally associated with good prognosis, its association with outcome seems to be influenced by other proteins such as Akt and PI3K. Acknowledgments Dena A Jerjees is funded by the higher committee of educational development in Iraq. Conflict of interest
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None.
Ethical standards This study was approved by the Nottingham Research Ethics Committee.
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