Conceived and designed the experiments: RF. Performed the experiments: MM VF PA GB SD SB. Analyzed the data: MM IS. Contributed reagents/materials/analysis tools: AF CN PV EM LP LR. Wrote the paper: RF. Other: Critical discussion: MM AS.
The authors have declared that no competing interests exist.
Tamoxifen is still the most widely used drug in hormone therapy for the treatment of breast cancer. Its benefits in adjuvant treatment are well documented in controlled and randomized clinical studies, which have demonstrated an increase in disease-free intervals of patients with positive hormonal receptors. However, the mechanisms involved in endocrine resistance are not clear. Laboratory and clinical data now indicate that bi-directional molecular cross-talk between nuclear or membrane ER and growth factor receptor pathways may be involved in endocrine resistance. We recently found a functional interaction between α6β4 integrin and ErbB-3 receptor to maintain the PI3K/Akt survival pathway of mammary tumour cells. We sought to improve understanding of this process in order to provide the involvement of both receptors insight into mechanism of Tamoxifen resistance.
Using human breast cancer cell lines displaying different levels of α6β4 and ErbB-3 receptors and a series of 232 breast cancer biopsies from patients submitted to adjuvant Tamoxifen monotherapy for five years, we evaluated the functional interaction between both receptors in relationship to Tamoxifen responsiveness. In mammary carcinoma cells, we evidenced that the α6β4 integrin strongly influence Akt phosphorylation through ErbB-3 protein regulation. Moreover, the ErbB-3 inactivation inhibits Akt phosphorylation, induces apoptosis and inhibits
We provided evidence that a strong relationship occurs between α6β4 and ErbB-3 positivity in ERβ1-negative breast cancers. We also found that the association between ErbB-3 and P-Akt positivity mainly occurs in ERβ1-negative breast cancer derived from patients with lower DFS indicating that both receptors are clinically relevant in predicting the response to Tamoxifen.
In many breast cancer (BC), activation of the phosphatidylinositol 3-kinase (PI3K) pathway may deeply reduce the efficacy to targeted therapies
BC remains one of the most heterogeneous tumors in terms of capability to give metastases, expression of hormone receptors and responsiveness to therapies and is the first cause of death for women aged 35–45 years
In the present work, using ER-positive human BC cell lines, we investigated the functional interaction between α6β4 and ErbB-3 proteins in relationship to TAM responsiveness. In addition, with the aim to translate our
We first evaluated the expression level of ERα and ERβ, β4 integrin subunit, ErbB2, and ErbB-3 in a series of human mammary tumor cell lines including MDA-MB 231, MDA-MB 361, SKBr3, BT474, BT549, and T47D. Analysis of ERα by Western blotting (
A. The expression of ERα was evaluated by western blot analysis. The anti-actin Ab was used to validate equivalent loading protein. B. ERβ1 expression was evaluated by RT-PCR from total mRNA extracted from the indicated cell lines using primers specific for human ERβ1 and the housekeeping aldolase genes. C. Mammary tumor cell lines MDA-MB 231, MDA-MB 361, SKBr3, BT474, BT549 and T47D were analyzed by FACS to reveal the expression level of β4 integrin subunit, ErbB-2 and ErbB-3 receptors.
Given that α6β4 integrin is the receptor for laminin 5 (LM5) and, as we previously demonstrated, ligation of the integrin to this substrate enhances PI3K signaling, we first verified the level of Akt phosphorylation upon stimulation in the mammary tumor cell lines. To this end, MDA-MB 361, BT474, SKBr3, BT549 and MDA-MB 231 cells were spread onto LM5 for 20 minutes and the level of Akt activity was evaluated by Ser473 phosphorylation. As reported in
A. BT549, MDA-MB 231, MDA-MB 361, BT474 and SKBr3 cells were serum-starved for 24 hrs and then the cells were spread onto LM5 and extracted in detergent. Equivalent amounts of protein were separated by SDS-PAGE and analyzed by immunobloting to evaluate the relative expression of β4 and phospho-Akt. Total-Akt Ab was used to validate equivalent loading of protein in each lane. B. MDA-MB 361, BT474 and SKBr3 cells were transiently transfected for 48 hrs with either scrambled or specific β4-shRNA and ErbB-3 siRNA. The cells were then serum-starved for 24 hrs and extracted in detergent. Equivalent amounts of protein were separated by SDS-PAGE and analyzed by immunobloting to evaluate the relative expression of β4, ErbB-3 and phospho-Akt. Hsp70 Ab was used to validate equivalent loading of protein in each lane.
To confirm the essential role of ErbB-3 protein in the activation of Akt by α6β4, a β4 shRNA (β4si) or an ErbB-3 siRNA (B3si) were expressed in MDA-MB 361, BT474 and SKBr3 cells, as previously described
To further evaluate the function of ErbB-3 in the PI3K survival pathway, we analyzed cell death and apoptosis in the absence of hormones and under TAM treatment of ErbB3 positive (SKBr3, MDA-MB 361, BT474 and T47D) and ErbB3 negative (MDAMB231) cell lines. As shown in
A. SKBr3, MDA-MB 361, BT474, T47D and MDAMB231 cells after three days of hormone deprivation were transiently transfected with either scrambled or specific ErbB-3 siRNA. Where specified, 24 hrs after transfection scrambled and ErbB3 interfered cells were pre-incubated for 24 hours at 37°C with TAM 2.5 µM. 48 hours following transfection, the cell death was evaluated by Trypan-blue exclusion. Statistical differences were evaluated by T test (p<0.05). B. Equivalent amounts of total cell lysate derived from the cell lines described in A were separated by SDS-PAGE and analyzed by immunobloting to evaluate the expression level of PARP cleavage. Hsp70 Ab was used to validate equivalent loading of protein in each lane. C. SKBr3, MDA-MB 361, BT474, T47D and MDAMB231 cells transfected as described in A were assayed for their ability to invade matrigel in the absence of hormone and under TAM treatment. Statistical differences were evaluated by T test (p<0.05).
It is widely reported that TAM resistance and, as a consequence, tumor progression may be also due to PI3K activation
To verify whether the functional interaction between α6β4 integrin and ErbB-3 receptor also occurred
A. Distribution (%) of the bio-pathological factors β4 integrin subunit, ErbB-3, P-Akt(ser473), ERβ1 and ErbB-2 in 232 TAM treated breast cancers. B. Representative immunohistochemically positive cases for β4, ErbB-3, P-Akt(ser473), ERβ1 and ErbB-2 protein detection and control tissue sections.
CHARACTERISTIC | % | |
Pre | 25 | 10.8 |
Post | 207 | 89.2 |
Invasive ductal carcinoma | 193 | 83.2 |
Invasive lobular carcinoma | 28 | 12.1 |
Tubular carcinoma | 7 | 3 |
Papillary carcinoma | 4 | 1.7 |
T1 | 176 | 79.9 |
T2 | 51 | 22 |
T3,T4 | 5 | 2.1 |
Negative | 193 | 83.2 |
Positive | 39 | 16.8 |
G1 | 53 | 22.8 |
G2 | 141 | 60.8 |
G3 | 38 | 16.4 |
Negative (≤10%) | 25 | 10.8 |
Positive (>10%) | 207 | 89.2 |
Negative (≤10%) | 55 | 23.7 |
Positive (>10%) | 177 | 76.3 |
range 39–95
Factors | Integrin β4 | ||
Neg and % | Low and High Pos and % | P | |
T1 | 45 (25.6) | 131 (74.4) | 0.48 |
T2-T4 | 17 (30.4) | 39 (69.6) | |
Negative | 48 (24.9) | 145 (75.1) | 0.16 |
Positive | 14 (35.9) | 25 (64.1) | |
G1 | 11 (20.8) | 42 (79.2) | 0.51 |
G2 | 41 (29.1) | 100 (70.9) | |
G3 | 10 (26.3) | 28 (73.7) | |
Negative | 51 (33) | 104 (67) | |
Positive | 11 (14) | 66 (86) | |
0.19 | |||
Negative | 30 (31.6) | 65 (68.4) | |
Low | 21 (22.8) | 56 (77.2) | |
High | 11 (18) | 49 (82) | |
Negative | 32 (43.2) | 42 (56.8) | |
Positive | 30 (19) | 128 (81) | |
Negative | 50 (28.9) | 123 (71.1) | 0.40 |
Positive | 12 (20.3) | 47 (79.7) |
χ Test
At a median follow up of 58 months (range 1–179 months), a total of 36 patients (15%) showed progressive disease.
The results of the univariate and multivariate analyses for DFS in the 232 patients included in this study are summarized in
Univariate analysis | Multivariate analysis | |||
Factors | HR (95% CI) | p value | HR (95% CI) | p value |
>2 cm | 2.42 (1.25–4.68) | 2.24 (1.10–4.53) | ||
G2 vs G1 | 3.19 (0.75–13.59) | 0.12 | 5.61 (1.25–25.30) | |
G3 vs G1 | 4.78 (1.05–21.69) | 10.23 (2.07–50.45) | ||
G2 | 0.67 (0.33–1.37) | 0.27 | 0.55 (0.26–1.17) | 0.12 |
N+ vs N0 | 2.25 (1.15–4.39) | n.s. | ||
positive | 1.37 (0.63–2.95) | 0.42 | n.s. | |
positive | 3.01 (1.56–5.82) | 2.16 (1.10–4.22) | ||
positive | 5.03 (1.90–13.32) | n.s | ||
negative | 3.88 (1.98–7.59) | 3.28 (1.56–6.87) | ||
positive | 1.12 (0.55–2.28) | 0.75 | n.s. |
Each variable that significantly affected DFS in the univariate analyses were introduced into a Cox proportional risk model. Multivariate analyses revealed that tumor size (HR 2.24, C.I. 1.10–4.53, p = 0.02), grading (G2 vs G1, HR 5.61, C.I. 1.25–25.30, p = 0.02 and G3 vs G1, HR 10.23, C.I. 2.07–50.45, p = 0.004), ErbB-3 expression (HR 2.16, C.I. 1.10–4.22, p = 0.024) and lack of ERβ1 (HR 3.28, C.I. 1.56–6.87, p = 0.002) were independent prognostic variables influencing DFS. ERβ1 negativity appears to be the most powerful prognostic indicator of a reduced DFS, indicating that ERβ1 positive tumors are more likely to be responsive to TAM therapy.
Kaplan-Meier curves (
Survival curves were generated according to the Kaplan-Meier method; statistical comparisons were made using the log-rank test.
On the basis of these results, we evaluated the impact on DFS of β4, ErbB-3, P-Akt and ERβ1 combination. β4 expression, even if associated to the other three variables, did not add further useful clinical information. In contrast, as shown in
Survival curves were generated according to the Kaplan-Meier method; statistical comparisons were made using the log-rank test.
It is widely known that α6β4 integrin expression and signaling are involved in the mechanisms that regulate tumor progression and resistance to apoptotic stimuli
Our results are of particular clinical interest, since the anti-estrogen TAM plays a central role in the treatment of human BC. Nevertheless, many tumors appear to be refractory to TAM, making it necessary to discover predictive markers that can accurately identify hormone responsive tumors. In this setting, till the discovery of ERβ, ERα was the single most informative marker, receptor-negative tumors rarely benefiting from endocrine therapy
Aimed at translating our in vitro results to human BC, we evaluated, by IHC, α6β4, ERβ1, ErbB3, and P-AKT expression in 232 primary mammary tumors derived from patients submitted to adjuvant TAM monotherapy. Even though we found a significant correlation between β4 and ErbB-3 expression and ERβ1 negativity, in the BCs we analyzed, the expression of the integrin did not influence the patient outcome.
ErbB-3 proteins mainly occurred in the P-Akt-positive and ERβ1-negative BC derived from patients with lower DFS. Although previous experimental studies have implied that α6β4 integrin facilitates tumor progression by regulating growth factor receptors signaling
The high percentage of mammary tumors we analyzed which over-express β4 integrin subunit is consistent with previous findings
These and numerous other studies conducted
From our data it is evident that ErbB-3 may represent a key molecule involved in the mechanisms of TAM resistance in ERβ1-negative BC. This finding is in agreement with a recent report demonstrating that ErbB-3 modulates ErbB-2-mediated proliferation, colony formation and resistance to TAM treatment
We can conclude that, even though the regulation of mammary tumor growth and survival by ERs and EGFR family members and the biology of β4 integrin in tumors are not completely known, our
The human mammary carcinoma cell lines, MDA-MB361, BT474, SKBr3, MDA-MB231, T47D and BT549 were obtained from the ATCC and maintained in DMEM medium supplemented with 10% FCS (INVITROGEN, Milan, Italy). Rat bladder epithelial cell line 804G was cultured in minimum essential medium supplemented with 10% FCS and employed for LM5 rich matrix preparation
The rat anti-hum β4 subunit (Clone 439-9B) was prepared as previously described and used in immunoprecipitation, and immunofluorescence (FACS) analysis experiments
The expression level of β4, ErbB2 and ErbB-3 in MDA-MB 231, MDA-MB 361, BT474, SKBr3, BT549 and T47D cells was detected by flow cytometry analysis of stained cells. In brief, cells harvested using citrate saline buffer (0.134 M KCl, 0.015 M Na citrate) were washed twice with cold PBS containing 0.002% EDTA and 10 mM NaN3 (washing buffer). Samples of 1×106 cells were incubated for 1 h at 4°C with saturating concentrations of primary antibodies diluted in PBS containing 0.5% bovine serum albumin (BSA). Cells were then washed three times with washing buffer (PBS containing 0.5% BSA) and incubated for 1 h at 4°C with 50 l of FITC-conjugated secondary antibodies [F(ab')2 (Cappel, West Chester, PA, U.S.A.)] diluted 1:20 in PBS/BSA. After three washes, the cells were suspended in 1 ml of washing buffer. Cell suspensions were analyzed by a flow-cytometer (Epics XL analyzer, Coulter Corporation, Miami, FL) after addition of 5l of a 1mg/ml solution of propidium iodide to exclude non-viable cells. At least 1×104 cells per sample were analyzed.
To analyze ER-α, β4 and ErbB-3 protein expression, the cells were lysed with RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P40, 0,1% deoxycholate, 0,1% SDS, 1mM PMSF, 5 mM Na3VO4, 50 mM protease inhibitors (SIGMA-Aldrich, Milan, IT) for 30 minutes at 4°C. Total cell lysates were clarified by centrifugation at 14,000 rpm for 30 minutes. Aliquots of cell extracts containing an equivalent amount of proteins were resolved by SDS-polyacrilamide gel electrophoresis 10% (SDS-PAGE) and transferred to nitrocellulose. To analyze Akt activation after stimulation by LM5, MDA-MB 361, BT474 and SKBr3 (1×106) cell lines, after serum starvation for 24 hours, were seeded onto 100 mm tissue culture dishes coated with LM5-rich matrix preparation from 804G cells. The cells were washed three times with ice cold PBS and lysed with NP40 buffer (1% Nonidet P40, 10% glycerol, 137 mM NaCl, 20 mM Tris HCl (pH 7,4), 50 mM NaF, 1 mM PMSF, 5mM Na3VO4, 50 mM protease inhibitors (SIGMA-Aldrich, Milan, IT) for 30 minutes at 4°C. Total cell lysates were clarified by centrifugation at 14,000 rpm for 30 minutes. Aliquots of cell extracts containing equivalent amounts of proteins were resolved by SDS-PAGE, transferred to nitrocellulose and probed with the rabbit polyclonal Ab directs to P-Akt. As secondary Abs, the horseradish peroxidase-coniugated goat anti-mouse or rabbit were used. The chemiluminescence was resolved by an enhanced chemiluminescence ECL kit (Amersham, Milan, IT). Total proteins were normalized by anti-actin, anti-Hsp70 and total-Akt Abs, respectively.
Total RNA was prepared using RNAzol B according to the manufacturer's procedure (Invitrogen, Milan, IT). Human ERβ1 mRNA for RT-PCR analysis was carried out using specific primers as previously described
hERb1 sense:
hERβ1 anti-sense:
The housekeeping aldolase mRNA was used as an internal control.
For the detection of ERβ by immunocytochemistry, 5×105 cells of each cell line (MDA-MB 231, MDA-MB 361, SKBr3, BT474, BT549 and T47D) were centrifuged onto glass slides (cytospin) and fixed in 2% formaldehyde for 10 minutes. Endogenous peroxidase was blocked by incubating in 3% H2O2 in PBS for 10 minutes. After two rinses in PBS, nonspecific binding was blocked by a 10-minute incubation with normal serum (ScyTek Laboratories, Logan, UT). Samples were then incubated in mouse anti-ERβ1 antibody (1∶20 dilution) in 0,5% bovine serum albumine with PBS overnight, in a humidified atmosphere. Detection steps were done using the UltraTek HRP kit according to the manufacturer's procedure (ScyTek Laboratories), and peroxidase activity was localized with DAB (diamino-benzidine) substrate. Slides were counterstained by Hematoxilin and mounted under a coverslip in glycerol.
The inactivation of β4 was obtained by the LipofectAMINE PLUS™ method (INVITROGEN) using pSUPER.retro vector containing β4-shRNA or scramble RNA (scr-shRNA) sequences. To inactivate ErbB-3 expression, cells were transiently transfected with Transit-TKO reagent (MIRUS, Medison, Wisconsin) following the manufacturer procedures with the ErbB-3 anti-sense double strand siRNA as previously described
SKBr3, MDA-MB 361, BT474, TD47D and MDA-MB 231 cells (3×105) were plated onto 60mm dishes in hormone-deprivation conditions for three days. The following day, the cells were trasiently transfected with a scrambled or ErbB-3 siRNA sequence and 24 hours after transfection the cells were treated with 2.5 µM TAM or ethanol as a control for 24 hrs. The viability of the cells was evaluated by Trypan blue exclusion. Each assay was repeated at least three times. Following the same procedure, the cells were lysed in Triton buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton x-100, 0.5% NP40, 2.5% sodium pyrophosphatate, 1 mM Na3VO4, 50 mM protease inhibitors) and sonicated for 15 seconds. Samples were boiled for 5 minutes at 95°C, resolved by SDS-polyacrilamide gel electrophoresis (8%), transferred to nitrocellulose and probed with a mouse anti-PARP Ab.
Chemoinvasion was assessed using a 48-well modified Boyden's chamber (NeuroProbe, Pleasanton, CA) and 8-µm pore polyvinyl pyrrolidone–free polycarbonate Nucleopore filters (Costar, New York, NY). The filters were coated with an even layer of 3 mg/mL Cultrex (Trevigen, Gaithersburg, MD). The lower compartment of the chamber was filled with 24 hours conditioned serum free medium produced from NIH3T3 fibroblasts. SKBr3, MDA MB361, BT474, TD47D and MDA-MB 231 cells, after 3 days of hormone deprivation, were plated (1.5×106 cells) onto 100 mm dishes. The following day, the cells were transfected with scrambled or ErbB-3 siRNA. Where specified, 24 hrs after transfection scrambled and ErbB-3 interferred cells were pre-incubated for 24 hours at 37°C with TAM 2.5 µM. The cells were, then, harvested (2×106 cells/ml) and placed in the upper compartment (45 µl/well) of the Boyden's chamber. After 8 hours of incubation at 37°C, the cells migrated on the lower surface of the filters were fixed and stained with DiffQuick (Merz-Dade, Dudingen, Switzerland). Then, the migrated cells in 12 high-power fields were counted. Each assay was carried out in quadruplicate and repeated at least three times. The ability of the cells to adhere to the filters was verified by staining the upper side of the filter for each cell line.
We studied a cohort of 232 hormonal receptor positive breast cancer patients surgically treated at the Regina Elena Cancer Institute (Rome, Italy) between 1986 and 2002, who had received an up-front adjuvant hormonal monotherapy with TAM at the dose of 20 mg per day for a maximum of 5 years. Invasive breast cancers were classified according to the World Health Organization Classification of Tumors
The study was reviewed and approved by the ethical committee of Regina Elena National Cancer Institute, and written informed consent was obtained from all patients.
β4 integrin, ERβ1, P-AKT(ser473), ErbB-2 and ErbB-3 expression were assessed by indirect immunoperoxidase staining. Immunohistochemical staining was carried out on 5-µm-thick paraffin-embedded tissues. Sections were harvested on SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany).
The deparaffinized and rehydrated sections were pretreated by microwave in 1mM citrate buffer (pH6.0) at 430 W (two 10′ cycles followed by a 5′ one) for ERβ1 and at 760 W (three 5′ cycles) for p-AKT(ser473), ErbB-2, ErbB-3 and β4 antigen.
Sections were incubated overnight with the anti-ERβ1 (clone PPG5/10, Biogenex,Space, Milan, Italy), the anti-β4 integrin (clone 450-11A directed to the cytoplasmic tail of the subunit)
The integrin β4 subunit was evidenced both in the membrane and in the cytoplasm of neoplastic cells and was scored considering both intensity and frequency from 0 to 2 according to the following criteria: 0. No Reaction, 1. Low Reaction (1–10% of positive cells with score +/++/+++ or >10–50% with score +), 2. High Reaction (>10–50% of positive cells with score ++/+++ or >50% with score +/++/+++). The P-AKT(ser473) immunostaining was scored as described for β4 protein.
The immunoreactions were revealed by a streptavidin-biotin-peroxidase system (Super Sensitive Link-Label IHC Detection System, Biogenex) using 3-amino-9-ethylcarbazole (Dako, Milan, IT) as a chromogenic substrate. All sections were slightly counterstained with Mayer's hematoxylin and mounted in aqueous mounting medium (UCS Diagnostics, Rome, IT). Evaluation of the immunohistochemical results was done independently and in blinded manner by two investigators (M.M, and P.A.).
The correlation between β4 integrin expression and the biopathological characteristic variables was tested by the Pearson Chi-Square test. For the purpose of our study, disease-free survival (DFS) was considered as a measure of poor outcome. The disease free survival was calculated from the date of tumor diagnosis to the date of first recurrence or metastasis. Patients without recurrence were censored at the time of last follow-up or death. The Hazard risk and the confidence limits were estimated for each variable using the Cox univariate model and adopting the most suitable prognostic category as the referent group. The DFS curves were estimated by the Kaplan-Meier product-limit method. The log-rank test was used to assess differences between subgroups, and significance was defined as p<0.05.
A multivariate Cox proportional hazard model was also developed using stepwise regression (forward selection) with predictive variables which were significant in the univariate analyses. The enter limit and remove limit were p = 0.10 and p = 0.15, respectively. The SPSS (11.0) statistical program was used for analysis.
The expression of ERbeta1 protein was evaluated by immunocytochemistry. 5×105 MDA-MB 231, MDA-MB 361, SKBr3, BT474, BT549 and T47D cells were centrifuged onto glass slides and fixed in 2% formaldehyde for the dectection of ERb1 expression.
(10.39 MB TIF)
Representative invading stained cells. Chemoinvasion was assessed using a 48-well modified Boyden's chamber and 8-μm pore polyvinyl pyrrolidone-free polycarbonate filters. SKBr3, MDA MB361, BT474, TD47D and MDA-MB 231 (src, scr/TAM, B3si, B3si/TAM) cells migrated on the lower surface of the filters were fixed and stained. Then, the migrated cells in 12 high-power fields were counted. Each assay was carried out in quadruplicate and repeated at least three times.
(10.34 MB TIF)
We would like to thank Silvia Soddu for helpful discussions and critical reading of the manuscript, Simona Nanni for helpful discussion in ERs field. Maria Pia Gentileschi for technical assistance, Maria Assunta Fonsi for the secretarial assistance and Michael Kenyon for his formal revision of the manuscript.