Abstract
The aim of this study is to compare the expression of autophagy related proteins in invasive lobular carcinoma (ILC) with that of autophagy related proteins in invasive ductal carcinoma (IDC), and to determinate its implication. Tissue microarray containing 114 ILC and 692 IDC was constructed, and immunohistochemistry was performed for autophagy related protein (beclin-1, LC3A, LC3B, p62) and Ki-67. No significant difference in expression of autophagy-related proteins between pleomorphic type (n = 12) and classic type (n = 102) of ILC was observed, whereas ILC and IDC showed distinguished features that tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 were highly expressed in IDC and tumoral BNIP3 was highly expressed in ILC (P < 0.001). Beclin-1 expression was correlated with ER negativity (P = 0.016) and TNBC type (P = 0.024). BNIP3 expression was correlated with ER positivity (p = 0.040). Using multivariate Cox analysis, shorter overall survival was associated with tumoral beclin-1 positivity (hazard ratio: 21.19, 95% CI: 1.098-409.1, P = 0.043). In conclusion, ILC and IDC showed different expression pattern of autophagy-related proteins in tumor and stroma that demonstrated by higher expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 in IDC, and higher expression of tumoral BNIP3 in ILC.
Keywords: Autophagy, breast cancer, lobular cancer
Introduction
Breast cancer is one of the most common cancers in women, accounts for about 23% of female carcinoma [1]. Invasive carcinoma of the breast is categorized into the several histologic subtypes, largely into invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) [1]. ILC comprises about 5-15% of invasive carcinoma [2,3], appears to increase more than IDC in recent decade, along with hormone replacement therapy and alcohol consumption [4,5]. ILC is distinguished from IDC by clinical and histological features. ILC usually presents as multiple lesions, and tends to involve bilateral breast [6,7]. Histologically, ILC is characterized by non-cohesive cancer cells lacking e-cadherin [8]. Metastasis sites of ILC include bone, gastrointestinal tract, uterus, meninges, and ovary, which are unusual pattern of involvement in case of IDC. Diffuse serosal involvement is often found in ILC [7,9,10].
Cancer cells survive under condition of oxygen and glucose deprivation with angiogenesis and/or aerobic glycolysis. If this regulatory compensation is insufficient to meet the high metabolic demand of highly aggressive malignant tumor, alternative metabolic pathway triggers autophagy, in which cells cannibalize and recycle their cytoplasmic components for energy production [11]. Although autophagy is believed to be important in tumor metabolism, expression of autophagy related protein in ILC is yet uncertain because most of prior studies have analyzed of IDC [12,13]. Since ILC is distinctive from IDC in clinical, histological, and molecular aspects, we assumed that status of autophagy related proteins are different from IDC. The aims of this study were to compare the expression of autophagy related proteins in ILC to IDC, and to determinate its implication.
Material and methods
Patient selection and clinicopathologic evaluation
Between January 2000 and December 2012, formalin-fixed paraffin-embedded (FFPE) tissue samples were collected at Severance Hospital, from patients that had resection of breast due to ILC. Tissue samples diagnosed of IDC, no specific type in 2006 were prepared for control group. The study was approved by the Institutional Review Board of Severance Hospital. Patients who had received preoperative chemotherapy were excluded. Histologic examination was performed by hematoxylin and eosin (H&E) staining. All the slides were reviewed retrospectively by breast pathologists (Koo JS). The histological grade was assessed using the Nottingham grading system [14]. Tumor staging was based on the 7th American Joint Committee on Cancer (AJCC) criteria. Disease-free survival (DFS) was calculated from the date of the first curative surgery to the date of the first loco-regional or systemic relapse, or death without any type of relapse. Overall survival (OS) was estimated from the date of the first curative operation to the date of the last follow-up or death from any cause. Clinicopathologic parameters evaluated in each breast cancer included patient age at initial diagnosis, lymph node metastasis, tumor recurrence, distant metastasis, and patient’s survival.
Tissue microarray
Through retrospective review of the H&E-stained slides, the most appropriate formalin-fixed paraffin-embedded (FFPE) tumor tissue samples were obtained. The most representative tumor areas were marked on FFPE blocks and then 3 mm tissue cores were extracted by punch machine from the selected areas. Extracted tumor spots were inserted in 6 x 5 recipient blocks. Every 2 tissue cores were extracted from each case for TMA construction.
Immunohistochemistry
The antibodies used for immunohistochemistry in this study are shown in Table 1. Three micro meter paraffin sections were deparaffinized and rehydrated by xylene and alcohol solution. Immunohistochemistry was performed using the Ventana Discovery XT automated stainer (Ventana Medical System, Tucson, AZ, USA). Antigen retrieval was performed using CC1 buffer (Cell Conditioning 1; citrate buffer pH 6.0, Ventana Medical System). Appropriate positive and negative controls for immunohistochemistry were included.
Table 1.
Clones, dilutions, and sources of antibodies used
| Antibody | Clone | Dilution | Source |
|---|---|---|---|
| Autophagy related | |||
| Beclin-1 | Polyclonal | 1:100 | Abcam, Cambridge, UK |
| LC3A | EP1528Y | 1:100 | Abcam, Cambridge, UK |
| LC3B | Polyclonal | 1:100 | Abcam, Cambridge, UK |
| p62 | SQSTM1 | 1:100 | Abcam, Cambridge, UK |
| BNIP3 | Ana40 | 1:100 | Abcam, Cambridge, UK |
| Molecular subtype-related | |||
| ER | SP1 | 1:100 | Thermo Scientific, CA, USA |
| PR | PgR | 1:50 | DAKO, Denmark |
| HER-2 | Polyclonal | 1:1500 | DAKO, Denmark |
| Ki-67 | MIB-1 | 1:150 | DAKO, Denmark |
Interpretation of immunohistochemical results
A cut-off value of 1% or more positively stained nuclei was used to define ER and AR positivity [15]. HER-2 staining was analyzed according to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines using the following categories: 0 = no immunostaining; 1+ = weak incomplete membranous staining, less than 10% of tumor cells; 2+ = complete membranous staining, either uniform or weak in at least 10% of tumor cells; and 3+ = uniform intense membranous staining in at least 30% of tumor cells [16]. HER-2 immunostaining was considered positive when strong (3+) membranous staining was observed whereas cases with 0 to 1+ were regarded as negative. The cases showing 2+ HER-2 expression were evaluated for HER-2 amplification by fluorescent in situ hybridization (FISH).
Interpretation of immunohistochemical staining for autophagy and redox-related proteins was determined by multiplying the proportion of stained cell (0% = 0, 1-29% = 1, 30-100% = 2) with the immunostaining intensity (negative = 0, weak = 1, moderate = 2, strong = 3). The final scores of 0-1, 2-4, and 5-6 were interpreted as negative, low positive, and high positive, respectively [17]. Ki-67 labeling indices (LI) were scored by counting the number of positively stained nuclei and expressed as a percentage of total tumor cells.
Tumor phenotype classification
In this study, we classified breast cancer phenotypes according to the immunohistochemistry results for ER, PR, HER-2, and Ki-67 LI. FISH results for HER-2 were as follows [18]: luminal A type: ER and/or PR positive, HER-2 negative, and Ki-67 LI < 14%; luminal B type: (HER-2 negative) ER and/or PR positive, HER-2 negative, and Ki-67 LI ≥ 14% and (HER-2 positive) ER and/or PR positive and HER-2 overexpressed and/or amplified; HER-2 type: ER and PR negative and HER-2 overexpressed and/or amplified; TNBC type: ER, PR, and HER-2 negative.
Statistical analysis
Data were statistically processed using SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL). Student’s t test and Fisher’s exact test were used for continuous and categorical variables, respectively. Statistical significance was assumed when P < 0.05. Kaplan-Meier survival curves and log-rank statistics were employed to evaluate time to tumor metastasis and time to survival. Multivariate regression analysis was performed using Cox proportional hazards model.
Results
Basal characteristics of ILC
Clinicopathologic characteristics of ILC are summarized in Table 2. Among 114 ILCs, 102 were classic type, and 12 were pleomorphic type. Pleomorphic type demonstrates older age (P = 0.033), higher nuclear grade (P < 0.001), higher histologic grade (P < 0.001), higher T stage (P = 0.026), PR negativity (P = 0.005), HER-2 positivity (P = 0.002), higher Ki-67 LI (P < 0.001), non-luminal A subtype (P < 0.001) than classic type.
Table 2.
Clinicopathologic characteristics of patients with invasive lobular carcinoma
| Parameter | Total N = 114(%) | Classic type N = 102 (%) | Pleomorphic type N = 12 (%) | P-value |
|---|---|---|---|---|
| Age (years) | 0.033 | |||
| < 50 | 63 (55.3) | 60 (58.8) | 3 (25.0) | |
| ≥ 50 | 51 (44.7) | 42 (41.2) | 9 (75.0) | |
| Nuclear grade | < 0.001 | |||
| 1/2 | 102 (89.5) | 102 (100.0) | 0 (0.0) | |
| 3 | 12 (10.5) | 0 (0.0) | 12 (100.0) | |
| Histologic grade | < 0.001 | |||
| I/II | 109 (95.6) | 102 (100.0) | 7 (58.3) | |
| III | 5 (4.4) | 0 (0.0) | 5 (41.7) | |
| T stage | 0.026 | |||
| T1 | 67 (58.8) | 64 (62.7) | 3 (25.0) | |
| T2/T3 | 47 (41.2) | 38 (37.3) | 9 (75.0) | |
| Lymph node metastasis | 0.749 | |||
| Absent | 80 (70.2) | 72 (70.6) | 8 (66.7) | |
| Present | 34 (29.8) | 30 (29.4) | 4 (33.3) | |
| ER | 0.158 | |||
| Negative | 7 (6.1) | 5 (4.9) | 2 (16.7) | |
| Positive | 107 (93.9) | 97 (95.1) | 10 (83.3) | |
| PR | 0.005 | |||
| Negative | 19 (16.7) | 13 (12.7) | 6 (50.0) | |
| Positive | 95 (83.3) | 89 (87.3) | 6 (50.0) | |
| HER-2 | 0.002 | |||
| Negative | 107 (93.9) | 99 (97.1) | 8 (66.7) | |
| Positive | 7 (6.1) | 3 (2.9) | 4 (33.3) | |
| Ki-67 LI | < 0.001 | |||
| ≤ 14 | 92 (80.7) | 88 (86.3) | 4 (33.3) | |
| > 14 | 22 (19.3) | 14 (13.7) | 8 (66.7) | |
| Molecular type | < 0.001 | |||
| Luminal A | 86 (75.4) | 83 (81.4) | 3 (25.0) | |
| Luminal B | 22 (19.3) | 15 (14.7) | 7 (58.3) | |
| HER-2 | 1 (0.9) | 0 (0.0) | 1 (8.3) | |
| TNBC | 5 (4.4) | 4 (3.9) | 1 (8.3) |
Expression of autophagy-related proteins in ILC according to the cytologic type
There was no difference in expression of autophagy related proteins in two subtypes of ILC. Beclin-1, LC3A, and p62 were absent in stromal cells in all cases (Table 3).
Table 3.
Expression of autophagy-related proteins in ILC according to cytologic type
| Parameter | Total N = 114 (%) | Classic type N = 102 (%) | Pleomorphic type N = 12 (%) | P-value |
|---|---|---|---|---|
| Beclin-1 (T) | 0.508 | |||
| Negative | 83 (72.8) | 73 (71.6) | 10 (83.3) | |
| Positive | 31 (27.2) | 29 (28.4) | 2 (16.7) | |
| LC3A (T) | 1.000 | |||
| Negative | 112 (98.2) | 100 (98.0) | 12 (100.0) | |
| Positive | 2 (1.8) | 2 (2.0) | 0 (0.0) | |
| LC3B (T) | 0.675 | |||
| Negative | 98 (86.0) | 88 (86.3) | 10 (83.3) | |
| Positive | 16 (14.0) | 14 (13.7) | 2 (16.7) | |
| LC3B (S) | 0.594 | |||
| Negative | 105 (92.1) | 93 (91.2) | 12 (100.0) | |
| Positive | 9 (7.9) | 9 (8.8) | 0 (0.0) | |
| p62 (T) | 0.220 | |||
| Negative | 68 (59.6) | 63 (61.8) | 5 (41.7) | |
| Positive | 46 (40.4) | 39 (38.2) | 7 (58.3) | |
| BNIP3 (T) | 0.210 | |||
| Negative | 38 (33.3) | 32 (31.4) | 6 (50.0) | |
| Positive | 76 (66.7) | 70 (68.6) | 6 (50.0) | |
| BNIP3 (S) | 1.000 | |||
| Negative | 113 (99.1) | 101 (99.0) | 12 (100.0) | |
| Positive | 1 (0.9) | 1 (1.0) | 0 (0.0) |
T: tumor; S: tumor stroma.
Comparison of the expression of autophagy-related proteins between IDC and ILC
ILC showed lower expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, and tumoral p62, and higher expression of tumoral BNIP3 than IDC (P < 0.001, Table 4 and Figure 1). Because molecular subtypes of ILC are largely composed of luminal type, IDC of luminal types were selected for further comparison. Different expression of autophagy related proteins was identified as above between ILC and IDC of luminal types (Table 5).
Table 4.
Expression of autophagy-related proteins in IDC and ILC
| Parameter | Total N = 806 (%) | IDC N = 692 (%) | ILC N = 114 (%) | P-value |
|---|---|---|---|---|
| Beclin-1 (T) | < 0.001 | |||
| Negative | 463 (57.4) | 380 (54.9) | 83 (72.8) | |
| Positive | 343 (42.6) | 312 (45.1) | 31 (27.2) | |
| LC3A (T) | 0.153 | |||
| Negative | 767 (95.2) | 655 (94.7) | 112 (98.2) | |
| Positive | 39 (4.8) | 37 (5.3) | 2 (1.8) | |
| LC3A (S) | < 0.001 | |||
| Negative | 588 (73.0) | 474 (68.5) | 114 (100.0) | |
| Positive | 218 (27.0) | 218 (31.5) | 0 (0.0) | |
| LC3B (T) | < 0.001 | |||
| Negative | 546 (67.7) | 448 (64.7) | 98 (86.0) | |
| Positive | 260 (32.3) | 244 (35.3) | 16 (14.0) | |
| LC3B (S) | < 0.001 | |||
| Negative | 556 (69.0) | 451 (65.2) | 105 (92.1) | |
| Positive | 250 (31.0) | 241 (34.8) | 9 (7.9) | |
| p62 (T) | < 0.001 | |||
| Negative | 325 (40.3) | 257 (37.1) | 68 (59.6) | |
| Positive | 481 (59.7) | 435 (62.9) | 46 (40.4) | |
| BNIP3 (T) | < 0.001 | |||
| Negative | 506 (62.8) | 468 (67.6) | 38 (33.3) | |
| Positive | 300 (37.2) | 224 (32.4) | 76 (66.7) | |
| BNIP3 (S) | 0.109 | |||
| Negative | 775 (96.2) | 662 (95.7) | 113 (99.1) | |
| Positive | 31 (3.8) | 30 (4.3) | 1 (0.9) |
T: tumor; S: tumor stroma.
Figure 1.
Comparison of expression of autophagy related proteins between invasive ductal carcinoma and invasive lobular carcinoma.
Table 5.
Expression of autophagy-related proteins in ILC and IDC
| Parameters | Total N = 566 (%) | IDC, luminal A and B type N = 452 (%) | ILC N = 114 (%) | P-value |
|---|---|---|---|---|
| Beclin-1 (T) | 0.005 | |||
| Negative | 346 (61.1) | 263 (58.2) | 83 (72.8) | |
| Positive | 220 (38.9) | 189 (41.8) | 31 (27.2) | |
| LC3A (T) | 0.348 | |||
| Negative | 560 (98.9) | 448 (99.1) | 112 (98.2) | |
| Positive | 6 (1.1) | 4 (0.9) | 2 (1.8) | |
| LC3A (S) | < 0.001 | |||
| Negative | 397 (70.1) | 283 (62.6) | 114 (100.0) | |
| Positive | 169 (29.9) | 169 (37.4) | 0 (0.0) | |
| LC3B (T) | < 0.001 | |||
| Negative | 402 (71.0) | 304 (67.3) | 98 (86.0) | |
| Positive | 164 (29.0) | 148 (32.7) | 16 (14.0) | |
| LC3B (S) | < 0.001 | |||
| Negative | 381 (67.3) | 276 (61.1) | 105 (92.1) | |
| Positive | 185 (32.7) | 176 (38.9) | 9 (7.9) | |
| p62 (T) | < 0.001 | |||
| Negative | 246 (43.5) | 178 (39.4) | 68 (59.6) | |
| Positive | 320 (56.5) | 274 (60.6) | 46 (40.4) | |
| BNIP3 (T) | < 0.001 | |||
| Negative | 347 (61.3) | 309 (68.4) | 38 (33.3) | |
| Positive | 219 (38.7) | 143 (31.6) | 76 (66.7) | |
| BNIP3 (S) | 0.143 | |||
| Negative | 548 (96.8) | 435 (96.2) | 113 (99.1) | |
| Positive | 18 (3.2) | 17 (3.8) | 1 (0.9) |
T: tumor; S: tumor stroma.
Correlation between autophagy-related proteins and clinicopathologic factors in ILC
In ILC, beclin-1 expression was correlated with ER negativity (P = 0.016), tumoral BNIP3 expression was correlated with ER positivity (P = 0.040). Expression of beclin-1 was variably expressed according to molecular subtypes, and was highly expressed in TNBC (P = 0.024, Figure 2).
Figure 2.
Correlation between the expression of autophagy-related proteins and clinicopathologic factors in invasive lobular carcinoma.
Impact of expression status for autophagy-related proteins on prognosis in ILC
Expression of autophagy related proteins in relation to prognosis in ILC were calculated using univariate analysis (Table 6). Although no significant autophagy related proteins were found associated with shorter DFS and shorter OS, tendency of shorter OS was revealed with tumoral beclin-1 positivity (P = 0.062, Figure 3). In multivariate Cox analysis to determine the independent predictors, higher histologic grade (I/II versus III, hazard ratio: 57.45, 95% CI: 5.162-639.5, P = 0.001) was significantly associated with shorter DFS, and pleomorphic type (hazard ratio: 38.96, 95% CI: 2.612-581.1, P = 0.008) and tumoral beclin-1 positivity (hazard ratio: 21.19, 95% CI: 1.098-409.1, P = 0.043) were significantly associated with shorter OS.
Table 6.
Univariate analysis using the log-rank test to determine the impact of autophagy-related protein expression in patients with invasive lobular carcinoma on disease-free survival and overall survival times
| Parameter | Disease-free survival | Overall survival | ||
|---|---|---|---|---|
|
| ||||
| 95% CI | P-value | 95% CI | P-value | |
| Beclin-1 (T) | n/a | 0.062 | ||
| Negative | n/a | 165 (161-170) | ||
| Positive | n/a | 84 (77-91) | ||
| LC3A (T) | n/a | n/a | ||
| Negative | n/a | n/a | ||
| Positive | n/a | n/a | ||
| LC3A (S) | n/a | n/a | ||
| Negative | n/a | n/a | ||
| Positive | n/a | n/a | ||
| LC3B (T) | n/a | 0.499 | ||
| Negative | n/a | 163 (157-169) | ||
| Positive | n/a | 85 (78-93) | ||
| LC3B (S) | n/a | n/a | ||
| Negative | n/a | n/a | ||
| Positive | n/a | n/a | ||
| p62 (T) | 0.853 | 0.954 | ||
| Negative | 163 (156-169) | 162 (155-169) | ||
| Positive | 93 (89-98) | 93 (89-97) | ||
| BNIP3 (T) | 0.246 | 0.281 | ||
| Negative | 159 (147-171) | 159 (147-171) | ||
| Positive | 88 (86-91) | 88 (86-91) | ||
| BNIP3 (S) | n/a | n/a | ||
| Negative | n/a | n/a | ||
| Positive | n/a | n/a | ||
T: tumor; S: tumor stroma.
Figure 3.
Overall survival according to the status of beclin-1 expression in invasive lobular carcinoma.
Discussion
We examined expression of autophagy related proteins in ILC, and compared it to those in IDC statistically. There was no difference in between the classic type and pleomorphic type of ILC. Pleomorphic type has been reported as to show higher histologic grade, ER negativity, PR negativity, HER-2 positivity, poor prognosis than classic type [19,20]. It has also been suspected that autophagy-related proteins were usually associated with high grade tumor and poor prognosis [21-25]. In IDC, expression of LC3A and LC3B were also correlated with histologic grade [12,13]. However, in present study no significant difference was identified between classic type and pleomorphic type. It might be due to the limitation of sample numbers of pleomorphic type, which was far less than numbers of classic type.
Autophagy-related proteins were differently expressed in ILD and IDC. ILC showed lower expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, and tumoral p62, and higher expression of tumoral BNIP3 than IDC (P < 0.001).
Although limited number of studies have been carried out about the difference in between ILC and IDC, autophagy-related proteins except BNIP3 were highly expressed in IDC rather than ILC. In general, higher proliferative activity of tumor cells induced increase of autophagy activity to meet the increased energy demand. IDC has been known to have higher Ki-67 LI than ILC [26], which was also confirmed in the present study. However, ILC demonstrated higher BNIP3 expression than IDC. BNIP3 is one of pro-apoptotic Bcl-2 members, induced by HIF-1α which is triggered in tumor microenvironment like hypoxia, leads to cell death by mitophagy [27]. In previous study, BNIP3 was expressed in normal breast tissues, lost in a significant portion of invasive cancers, which was correlated with poor prognostic features like positive lymph node status and higher mitotic activity index [28]. Higher expression of BNIP3 in ILC than IDC in present study is compatible with prior studies, as Ki-67 LI is lower in ILC, which is also correlated with the loss of BNIP3 in tumor with higher Ki-67 LI.
In the current study, divergent expression of stromal autophagy-related protein between IDC and ILC were observed. LC3A was expressed higher in IDC than ILC. Autophagy-related proteins in tumor stroma has been also been reported in the previous studies [12]. In the breast cancer, autophagy activity in the tumor stroma is explained by reverse Warburg effect, which postulates the metabolic interaction between tumor cell and stromal cell in the breast cancer. Glycolysis, mitochondrial dysfunction, increased autophagy occur in stromal cells by reactive oxygen species released from tumor cells. Tumor cells generate ATP through oxidative phosphorylation, by modifying the lactate produced from glycolysis of stromal cells [29-32]. Stromal cell with increased autophagy activity is defined as cancer-associated fibroblast (CAF), which is characterized by loss of caveolin-1 [31]. Contrary to the stroma in IDC which demonstrates variable histologic features such as desmoplasia and fibrosis, no distinctive alteration is seen in the stroma of ILC. Only subset of breast cancer shows reverse Warburg effect as mentioned in prior study that 15% of tumor stroma in breast cancer showed loss of caveolin-1 [33]. However, this phenomenon in ILC is yet elucidated. In present study, autophagy-related proteins were differently expressed in stroma of IDC and ILC. Further analysis of this different signature of stroma in ILC is required whether CAF and metabolic interaction between tumor cell and stroma are associated in ILC.
We found beclin-1 positivity as independent poor prognostic factor, which was concordant with prior findings of correlation between beclin-1 expression and poor prognosis in ovary cancer [34], stomach cancer [35], larynx cancer [36], and uterine endometrial cancer [37]. However, it is controversial because other studies have demonstrated decreased of low expression of beclin-1 was associated with poor prognosis in cancer [38-40]. It is suspected that this controversy could be originated from dual role of autophagy that regulates both tumor survival and tumor suppression. Further validation in ILC is needed.
In present study, autophagy-related proteins were found in both tumor cell and stroma of ILC. Based on this result, inhibitor of autophagy could allow the target treatment in ILC. To date, as autophagy inhibitor has been discovered as suppressor of diverse tumors [41-44], further exploring of ILC is also required.
In conclusion, the present study analyzed expression pattern of autophagy-related proteins between IDC and ILC that demonstrated by higher expression of tumoral beclin-1, stromal LC3A, tumoral LC3B, tumoral p62 in IDC, and higher expression of tumoral BNIP3 in ILC.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1002886).
Disclosure of conflict of interest
None.
References
- 1.Tavassoli FA, Devilee P. Pathology and genetics of tumours of the breast and female genital organs. Lyon: IAPS Press; 2003. International Agency for Research on Cancer and World Health Organization. [Google Scholar]
- 2.Li CI, Anderson BO, Daling JR, Moe RE. Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA. 2003;289:1421–1424. doi: 10.1001/jama.289.11.1421. [DOI] [PubMed] [Google Scholar]
- 3.Li CI, Uribe DJ, Daling JR. Clinical characteristics of different histologic types of breast cancer. Br J Cancer. 2005;93:1046–1052. doi: 10.1038/sj.bjc.6602787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Li CI, Chlebowski RT, Freiberg M, Johnson KC, Kuller L, Lane D, Lessin L, O’Sullivan MJ, Wactawski-Wende J, Yasmeen S, Prentice R. Alcohol consumption and risk of postmenopausal breast cancer by subtype: the women’s health initiative observational study. J Natl Cancer Inst. 2010;102:1422–1431. doi: 10.1093/jnci/djq316. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Reeves GK, Beral V, Green J, Gathani T, Bull D. Hormonal therapy for menopause and breast-cancer risk by histological type: a cohort study and meta-analysis. Lancet Oncol. 2006;7:910–918. doi: 10.1016/S1470-2045(06)70911-1. [DOI] [PubMed] [Google Scholar]
- 6.Debnath J, Baehrecke EH, Kroemer G. Does autophagy contribute to cell death? Autophagy. 2005;1:66–74. doi: 10.4161/auto.1.2.1738. [DOI] [PubMed] [Google Scholar]
- 7.Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi Y, Gelinas C, Fan Y, Nelson DA, Jin S, White E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006;10:51–64. doi: 10.1016/j.ccr.2006.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.De Leeuw WJ, Berx G, Vos CB, Peterse JL, Van de Vijver MJ, Litvinov S, Van Roy F, Cornelisse CJ, Cleton-Jansen AM. Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ. J Pathol. 1997;183:404–411. doi: 10.1002/(SICI)1096-9896(199712)183:4<404::AID-PATH1148>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
- 9.Sastre-Garau X, Jouve M, Asselain B, Vincent-Salomon A, Beuzeboc P, Dorval T, Durand JC, Fourquet A, Pouillart P. Infiltrating lobular carcinoma of the breast. Clinicopathologic analysis of 975 cases with reference to data on conservative therapy and metastatic patterns. Cancer. 1996;77:113–120. doi: 10.1002/(SICI)1097-0142(19960101)77:1<113::AID-CNCR19>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
- 10.Lamovec J, Bracko M. Metastatic pattern of infiltrating lobular carcinoma of the breast: an autopsy study. J Surg Oncol. 1991;48:28–33. doi: 10.1002/jso.2930480106. [DOI] [PubMed] [Google Scholar]
- 11.Mizushima N. Autophagy: process and function. Genes Dev. 2007;21:2861–2873. doi: 10.1101/gad.1599207. [DOI] [PubMed] [Google Scholar]
- 12.Choi J, Jung W, Koo JS. Expression of autophagy-related markers beclin-1, light chain 3A, light chain 3B and p62 according to the molecular subtype of breast cancer. Histopathology. 2013;62:275–286. doi: 10.1111/his.12002. [DOI] [PubMed] [Google Scholar]
- 13.Sivridis E, Koukourakis MI, Zois CE, Ledaki I, Ferguson DJ, Harris AL, Gatter KC, Giatromanolaki A. LC3A-positive light microscopy detected patterns of autophagy and prognosis in operable breast carcinomas. Am J Pathol. 2010;176:2477–2489. doi: 10.2353/ajpath.2010.090049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19:403–410. doi: 10.1111/j.1365-2559.1991.tb00229.x. [DOI] [PubMed] [Google Scholar]
- 15.Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J. Clin. Oncol. 2010;28:2784–2795. doi: 10.1200/JCO.2009.25.6529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, Wheeler TM, Hayes DF. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J. Clin. Oncol. 2007;25:118–145. doi: 10.1200/JCO.2006.09.2775. [DOI] [PubMed] [Google Scholar]
- 17.Won KY, Kim GY, Kim YW, Song JY, Lim SJ. Clinicopathologic correlation of beclin-1 and bcl-2 expression in human breast cancer. Hum Pathol. 2010;41:107–112. doi: 10.1016/j.humpath.2009.07.006. [DOI] [PubMed] [Google Scholar]
- 18.Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ. Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol. 2011;22:1736–1747. doi: 10.1093/annonc/mdr304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Choi J, Jung WH, Koo JS. Metabolism-related proteins are differentially expressed according to the molecular subtype of invasive breast cancer defined by surrogate immunohistochemistry. Pathobiology. 2013;80:41–52. doi: 10.1159/000339513. [DOI] [PubMed] [Google Scholar]
- 20.Pinheiro C, Sousa B, Albergaria A, Paredes J, Dufloth R, Vieira D, Schmitt F, Baltazar F. GLUT1 and CAIX expression profiles in breast cancer correlate with adverse prognostic factors and MCT1 overexpression. Histol Histopathol. 2011;26:1279–1286. doi: 10.14670/HH-26.1279. [DOI] [PubMed] [Google Scholar]
- 21.Karpathiou G, Sivridis E, Koukourakis MI, Mikroulis D, Bouros D, Froudarakis ME, Giatromanolaki A. Light-chain 3A autophagic activity and prognostic significance in non-small cell lung carcinomas. Chest. 2011;140:127–134. doi: 10.1378/chest.10-1831. [DOI] [PubMed] [Google Scholar]
- 22.Pirtoli L, Cevenini G, Tini P, Vannini M, Oliveri G, Marsili S, Mourmouras V, Rubino G, Miracco C. The prognostic role of Beclin 1 protein expression in high-grade gliomas. Autophagy. 2009;5:930–936. doi: 10.4161/auto.5.7.9227. [DOI] [PubMed] [Google Scholar]
- 23.Sivridis E, Koukourakis MI, Mendrinos SE, Karpouzis A, Fiska A, Kouskoukis C, Giatromanolaki A. Beclin-1 and LC3A expression in cutaneous malignant melanomas: a biphasic survival pattern for beclin-1. Melanoma Res. 2011;21:188–195. doi: 10.1097/CMR.0b013e328346612c. [DOI] [PubMed] [Google Scholar]
- 24.Spowart JE, Townsend KN, Huwait H, Eshragh S, West NR, Ries JN, Kalloger S, Anglesio M, Gorski SM, Watson PH, Gilks CB, Huntsman DG, Lum JJ. The autophagy protein LC3A correlates with hypoxia and is a prognostic marker of patient survival in clear cell ovarian cancer. J Pathol. 2012;228:437–447. doi: 10.1002/path.4090. [DOI] [PubMed] [Google Scholar]
- 25.Wan XB, Fan XJ, Chen MY, Xiang J, Huang PY, Guo L, Wu XY, Xu J, Long ZJ, Zhao Y, Zhou WH, Mai HQ, Liu Q, Hong MH. Elevated Beclin 1 expression is correlated with HIF-1alpha in predicting poor prognosis of nasopharyngeal carcinoma. Autophagy. 2010;6:395–404. doi: 10.4161/auto.6.3.11303. [DOI] [PubMed] [Google Scholar]
- 26.Frolik D, Caduff R, Varga Z. Pleomorphic lobular carcinoma of the breast: its cell kinetics, expression of oncogenes and tumour suppressor genes compared with invasive ductal carcinomas and classical infiltrating lobular carcinomas. Histopathology. 2001;39:503–513. doi: 10.1046/j.1365-2559.2001.01252.x. [DOI] [PubMed] [Google Scholar]
- 27.Burton TR, Gibson SB. The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death. Cell Death Differ. 2009;16:515–523. doi: 10.1038/cdd.2008.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Koop EA, van Laar T, van Wichen DF, de Weger RA, Wall E, van Diest PJ. Expression of BNIP3 in invasive breast cancer: correlations with the hypoxic response and clinicopathological features. BMC Cancer. 2009;9:175. doi: 10.1186/1471-2407-9-175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Bonuccelli G, Tsirigos A, Whitaker-Menezes D, Pavlides S, Pestell RG, Chiavarina B, Frank PG, Flomenberg N, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Ketones and lactate “fuel” tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism. Cell Cycle. 2010;9:3506–3514. doi: 10.4161/cc.9.17.12731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Martinez-Outschoorn UE, Balliet RM, Rivadeneira DB, Chiavarina B, Pavlides S, Wang C, Whitaker-Menezes D, Daumer KM, Lin Z, Witkiewicz AK, Flomenberg N, Howell A, Pestell RG, Knudsen ES, Sotgia F, Lisanti MP. Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells. Cell Cycle. 2010;9:3256–3276. doi: 10.4161/cc.9.16.12553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank PG, Casimiro MC, Wang C, Fortina P, Addya S, Pestell RG, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation. Cell Cycle. 2010;9:2201–2219. doi: 10.4161/cc.9.11.11848. [DOI] [PubMed] [Google Scholar]
- 32.Pavlides S, Whitaker-Menezes D, Castello-Cros R, Flomenberg N, Witkiewicz AK, Frank PG, Casimiro MC, Wang C, Fortina P, Addya S, Pestell RG, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma. Cell Cycle. 2009;8:3984–4001. doi: 10.4161/cc.8.23.10238. [DOI] [PubMed] [Google Scholar]
- 33.Koo JS, Park S, Kim SI, Lee S, Park BW. The impact of caveolin protein expression in tumor stroma on prognosis of breast cancer. Tumour Biol. 2011;32:787–799. doi: 10.1007/s13277-011-0181-6. [DOI] [PubMed] [Google Scholar]
- 34.Zhao Y, Chen S, Gou WF, Xiao LJ, Takano Y, Zheng HC. Aberrant Beclin 1 expression is closely linked to carcinogenesis, differentiation, progression, and prognosis of ovarian epithelial carcinoma. Tumour Biol. 2014;35:1955–64. doi: 10.1007/s13277-013-1261-6. [DOI] [PubMed] [Google Scholar]
- 35.Yu M, Gou WF, Zhao S, Xiao LJ, Mao XY, Xing YN, Takahashi H, Takano Y, Zheng HC. Beclin 1 expression is an independent prognostic factor for gastric carcinomas. Tumour Biol. 2013;34:1071–1083. doi: 10.1007/s13277-013-0648-8. [DOI] [PubMed] [Google Scholar]
- 36.Huang L, Wang S, Li SS, Yang XM. Prognostic significance of Beclin-1 expression in laryngeal squamous cell carcinoma. Pathol Oncol Res. 2013;19:771–777. doi: 10.1007/s12253-013-9642-0. [DOI] [PubMed] [Google Scholar]
- 37.Giatromanolaki A, Koukourakis MI, Koutsopoulos A, Chloropoulou P, Liberis V, Sivridis E. High Beclin 1 expression defines a poor prognosis in endometrial adenocarcinomas. Gynecol Oncol. 2011;123:147–151. doi: 10.1016/j.ygyno.2011.06.023. [DOI] [PubMed] [Google Scholar]
- 38.Dong M, Wan XB, Yuan ZY, Wei L, Fan XJ, Wang TT, Lv YC, Li X, Chen ZH, Chen J, Lin Q, Wen JY, Ma XK, Liu Q, Wu XY. Low expression of Beclin 1 and elevated expression of HIF-1alpha refine distant metastasis risk and predict poor prognosis of ER-positive, HER2-negative breast cancer. Med Oncol. 2013;30:355. doi: 10.1007/s12032-012-0355-0. [DOI] [PubMed] [Google Scholar]
- 39.Lin HX, Qiu HJ, Zeng F, Rao HL, Yang GF, Kung HF, Zhu XF, Zeng YX, Cai MY, Xie D. Decreased expression of Beclin 1 correlates closely with Bcl-xL expression and poor prognosis of ovarian carcinoma. PLoS One. 2013;8:e60516. doi: 10.1371/journal.pone.0060516. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Liu GH, Zhong Q, Ye YL, Wang HB, Hu LJ, Qin ZK, Zeng MS, Zeng BH. Expression of beclin 1 in bladder cancer and its clinical significance. Int J Biol Markers. 2013;28:56–62. doi: 10.5301/JBM.2012.9769. [DOI] [PubMed] [Google Scholar]
- 41.Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A, Thompson CB. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007;117:326–336. doi: 10.1172/JCI28833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Carew JS, Medina EC, Esquivel JA 2nd, Mahalingam D, Swords R, Kelly K, Zhang H, Huang P, Mita AC, Mita MM, Giles FJ, Nawrocki ST. Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. J Cell Mol Med. 2010;14:2448–2459. doi: 10.1111/j.1582-4934.2009.00832.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Carew JS, Nawrocki ST, Kahue CN, Zhang H, Yang C, Chung L, Houghton JA, Huang P, Giles FJ, Cleveland JL. Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance. Blood. 2007;110:313–322. doi: 10.1182/blood-2006-10-050260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Gupta A, Roy S, Lazar AJ, Wang WL, McAuliffe JC, Reynoso D, McMahon J, Taguchi T, Floris G, Debiec-Rychter M, Schoffski P, Trent JA, Debnath J, Rubin BP. Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor (GIST) Proc Natl Acad Sci U S A. 2010;107:14333–14338. doi: 10.1073/pnas.1000248107. [DOI] [PMC free article] [PubMed] [Google Scholar]