Samples

Tumoral (n = 90) and adjacent (n = 40) tissues were obtained from women over 45 years old with BC (Table 1). All women included in the present study (>45 years old; patients without treatment) were born in Mexico with grandparents also born in Mexico and from families with no history of cancer (de novo tumors). All tumor cases were histologically reviewed by at least two pathologists, one of them selected and marked representative tumor and adjacent areas for the preparation of TMA. The ethical committee of the National Research Council of Instituto Mexicano del Seguro Social approved the study and thus meets the standards of the Declaration of Helsinki in its revised version of 1975 and its amendments of 1983, 1989, and 1996 (JAMA 1997;277:925-926). Patients were informed about the study and ask to sign an informed consent letter.

BC TMA and IHC

TMAs were prepared from blocks of formalin-fixed paraffin, embedded BC specimens obtained from two Mexican hospitals (Luis Castelazo Gynecology Hospital and Oncology Hospital, CMNSXXI, IMSS). Tissue (2 µm thick) was cored from representative areas of tumor and adjacent tissues for TMA. Clinical and pathologic data were obtained from medical and pathology records. Sections 3 µm thick in size were cut for routine hematoxylin and eosin (H&E) staining and IHC. Microarrays were processed in a Ventana Benchmark system (Roche). TMAs were dehydrated using ascending alcohol solutions, cleared in a xylol solution, and mounted in a synthetic resin. The primary antibodies used were anti-BIK (Abcam, ab52182) or its phosphorylated form (anti-p-BIK) (Abcam, ab55179). A set of microarrays was reacted with normal rabbit IgG, as a negative control. Then, microarrays were observed in a DM750 Leica microscope (Wetzlar, Germany), and digital images obtained using the LEZ software. Two independent observers evaluated the arrays. Densitometry was performed using the Image-Pro software (Media Cybernetics, Rockville, USA) and graded as high (~10,000-15,000 pixels), intermediate (~2000 pixels), and low signal (10-50 pixels), according to the intensity of staining present in tissues.

Densitometry analysis

Densitometry was performed using the Image-Pro software (Media Cybernetics, Rockville, USA) selecting: measure --- Count/Size --- Measure --- Select Measurements --- Density (mean). According to the signal present in tissues, density was divided into high (~10,000-15,000 pixels), intermediate (~2,000 pixels), and low (10-50 pixels) signal; it is important to mention that low signal was not considered and it was taken as background. The background signal was the one obtained from the negative controls (tissue incubation without the specificantibody [anti-BIK]), and it was subtracted from the specific signal for BIK. Furthermore, when the signal was very close to zero (low signal: ~10-50 pixels), it was subtracted from the specific one.

Data represent mean ± SEM from three experiments in triplicated; statistical significance was determined by the Student's t-test (p < 0.05).

Cell culture

Cell lines HMEC, MCF-7, MCF-10A, and MDA-MB-231 (American Type Culture Collection, ATTC, USA) were maintained in Dulbecco's Modified Eagles Medium F:12 (DMEM; Invitrogen, Carlsbad, CA, USA) at 37° C in 5% CO₂ atmosphere. For HMEC and MCF-10A cells, the medium was supplemented with 5% (v/v) horse serum + 10 µg/mL human insulin + 20 ng/mL hEGF + 100 ng/mL cholera toxin + 0.5 µg/mL hydrocortisone. Meanwhile, MCF-7 and MDA-MB-231 cell lines were maintained in the presence of 10% (v/v) fetal bovine serum-containing penicillin (100 U/mL) and streptomycin (100 µg/mL).

qPCR

qPCR for BIK was done in non-tumorigenic cell lines HMEC and MCF10A, and in the BC cells MCF-7 (receptor positive) and MDA-MB-231 (triple negative, claudin-low), by following the conditions established by Pfaffl⁹. Gene 18S was amplified, purified, and quantified. Serial dilutions (1:200; 1:20,000; 1:2,000,000; 1:20,000,000; and 1:20,000,000,000) were used to construct the standard curve. BIK amplification was always carried out in the presence of the standard curve. Primers used for 18S amplification: forward: 5´-TACCACATCCAAGGAAGGCAGCA-3´; reverse: 5´-TGG AATTACCGCGGCTTGCTGGCA-3´. Primers used for BIK: forward: 5´-CCCCGAGATAGTGCTGGAAC-3´; reverse: 5´-GCCGAGGGCATCACATATCA-3´. Statistics analysis (p < 0.05; Student's t-test) and graphical visualization were carried out with GraphPad Prism 5 (San Diego, CA).

Western blot

Western blot was performed as previously described¹⁰. Cell cultures lysed in RIPA-Tris buffer (mM: EGTA 2; NaCl 316; NaF 50; Tris-HCl 20; Na3VO4 100, PMSF 100, and EDTA 100; 0.1% of leupeptin and aprotinin; sodium dodecyl sulfate [SDS] 0.2% and Triton-X 100 2%) under constant shaking for 2 h at 4°C. After that, the sample was centrifuged for 5 min at 20,800 g and the supernatant (30 µg of protein) was denatured in Laemmli sample buffer, resolved through 15% SDS-polyacrylamide gels and electroblotted to polyvinylidene difluoride membranes. Membranes were incubated for 90 min in Tris-buffered saline (TBS) containing 5% dried skimmed milk and 0.1% Tween 20 (TBS-Tween milk) to block the non-specific protein-binding sites. Subsequently, membranes were incubated for 14 h at 4°C with primary antibodies (anti-BIK [Abcam ab52182]; anti-pBIK [Abcam ab55179]; or anti-GAPDH [EMD Millipore ABS16]) and subsequently with the secondary antibody (ECL-anti-rabbit [Abcam ab6721]) diluted in TBS-Tween milk. The protein was detected using an ECL Western blot detection kit (Millipore). The blots were subjected to a densitometry analysis and data were analyzed using the GraphPad Prism 5 software (San Diego, CA). The intensity values for BIK were normalized against the GAPDH loading control. Data were the mean ± SEM from three independent experiments (p < 0.05; Student's t-test).

BIK expression

To establish the expression of BIK on different types and grades of BC tissues, IHC studies were performed on TMA. Invasive ductal carcinoma (IDC) showed an overexpression of BIK in Grade I (142 ± 34%; P < 0.04; Student's t-test vs. control [I]) and Grades IIA (226 ± 37%; p < 0.0007; Student's t-test vs. control [IIA]) and IIB (521 ± 68%; p < 0.007; Student's t-test vs. control [IIB]) relative to adjacent tissues (Fig. 1A). Similarly, lobular carcinoma (LC) Grades IIA and IIB had a considerable BIK signal increase by 293 ± 53% (p < 0.02; Student's t-test vs. control [IIA]) and 347 ± 86% (p < 0.007; Student's t-test vs. control [IIB]) when compared to the adjacent tissue (Fig. 1B).

Figure 1. A-D BIK expression in breast cancer microarrays. Both IDC and lobular carcinoma (LC) showed increased BIK expression levels. By contrast, BIK was down-expressed in canalicular carcinoma and phyllodes tumors. Data represent the mean ± SEM in triplicated. *(p < 0.04; Student's t-test vs. control [I]); **(p < 0.007; Student's t-test vs. control [IIB]); ***(p < 0.0007; Student's t-test vs. control [IIA]); ^φ(p < 0.01; Student's t-test vs. control [I]); ^κ(p < 0.017; Student's t-test vs. control [IIB]); ^γ(p < 0.02; Student's t-test vs. control [IIA]). 

Carcinoma canalicular Grades I and IIB expressed lower levels of BIK than their adjacent tissues. The decrease observed was of 71 ± 12% (p < 0.01; Student's t-test vs. control [I]) for Grade I and by 97 ± 0.53% (p < 0.017; Student's t-test vs. control [IIB]) for Grade IIB (Fig. 1C). Similarly, rare breast tumor phyllodes Grades IIA and IIB expressed lower BIK levels −61 ± 12% (p < 0.02; Student's t-test vs. control [IIA]) and −59 ± 14% (p < 0.02; Student's t-test vs. control [IIB]) than their adjacent tissues, respectively (Fig. 1D).

p-BIK expression

Similar to BIK expression levels, the phosphorylated form of BIK "p-BIK" (Thr-33) gradually increased in Grades I (72 ± 22%; p < 0.001; Student's t-test vs. control [I]), IIA (168 ± 24%; p < 0.0001; Student's t-test vs. control [IIA]), and IIB (256 ± 26%; p < 0.0001; Student's t-test vs. control [IIB]) of IDC, relative to adjacent tissues (Fig. 2A). By contrast, p-BIK expression did not differ from adjacent tissues and tumor samples and between distinct tumor grades of LC (Fig. 2B). In the same way, p-BIK did not exhibit expression differences in intraductal carcinoma and mixed ductal and LC (Fig. 2C and D).

Figure 2. A-D p-BIK expression in breast cancer microarrays. The phosphorylated form of BIK showed expression changes in invasive ductal carcinoma, but it was not statistically different in the other tumor subtypes. Values are the mean ± SEM performed in triplicated. *(p < 0.001; Student's t-test vs. control [I]); ***(p < 0.0001; Student's t-test vs. control). 

BIK expression in BC cell lines

The expression of BIK was tested at mRNA and protein levels in the HMEC, MCF10A, MCF-7, and MDA-MB-231 cell lines. At mRNA level, HMEC (0.17 ± 0.04 fg) and MDA-MB-231 (0.14 ± 0.0023 fg) cells showed higher mRNA expression levels than those observed for MCF-10A (1.9 × 10⁻⁵ ± 6.3 × 10⁻⁸ fg) and MCF-7 (0.002 ± 0.00092 fg) cell lines (Fig. 3A). In a similar manner, Western blot analysis showed that BIK expression was higher in MDA-MB-231 (67 ± 4%) than in MCF-7 cells (Fig. 3B).

Figure 3 A: BIK expression in BC cell lines. BIK mRNA levels were determined by quantitative polymerase chain reaction (qPCR) - using a standard curve (RNA, 18S ribosomal gene) - and B: Western blot. qPCR values are the mean ± SEM of three experiments performed in triplicated. *(p < 0.02; Student's t-test vs. human mammary epithelial cells [HMEC] or MDA-MB-231); **(p < 0.0002; Student's t-test vs. HMEC or MDA-MB-231). Western blot data represent the mean ± SEM of three independent experiments. ***(p < 0.0001; Student's t-test vs. MCF-7).