Patients and clinical samples
The experiments were carried out using archival paraffin blocks of invasive ductal breast carcinomas (n = 495) and fibrocystic breast disease (FBD) – mastopathy (n = 38). Samples were obtained during surgical resection between 2010 and 2016 at the Polish Mother Memorial Hospital Research Institute in Lodz. Paraffin sections of the IDC and FBD samples were stained with hematoxylin and eosin (H&E) to verify the appropriateness of immunohistochemical (IHC) analyses. Clinical data were derived from hospital archives and are summarized in Table 1.
Table 1. Clinicopathological data of the studied cases of invasive ductal breast carcinoma.
Characteristic
|
No. (%) of patients (n = 495)
|
Tumour size
T1 (< 2 cm)
T2 (2-5 cm)
T3 (> 5 cm)
T4
|
190 (38.0)
129 (26.0)
130 (26.0)
46 (9.0)
|
Tumour stage - pT
1
2
3
4
Grading
I
II
III
Nodal status pN
N0
N1
N2
N3
ER status
Positive
Negative
PR status
Positive
Negative
HER2 status
Positive
Negative
Triple negative status
Positive
Negative
|
313 (63.23)
152 (30.71)
19 (3.84)
11 (2.22)
92 (18.59)
312 (63.03)
91 (18.38)
298 (60.20)
167 (33.74)
18 (3.64)
12 (2.42)
324 (65.45)
171 (34.55)
312 (63.03)
183 (36.97)
311 (62.83)
184 (37.17)
40 (8.08)
455 (91.92)
|
|
|
Histopathological analysis of IHC reactions
KAT6A and KAT6B immunocytochemical expression was established using the ImmunoReactive Score (IRS) according to Remmele and Stenger [54]. This scale considers both the intensity of the color (staining) of the reaction and the percentage of positively stained cells. The final score, which represents the product (multiplication) of these two parameters, ranged from 0 to 12. Statistical analysis was performed using the Mann-Whitney U-test, ANOVA Kruskal-Wallis test, and Spearman test. Differences were considered statistically significant at p < 0.05.
Cell lines
For our studies, the breast carcinoma cell lines MCF-7, T-47D, and MDA-MB-231 (obtained from American Type Culture Collection ATCC, Manassas, VA, USA), SK-BR-3, BT-474 (from the Cell Lines Collection of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Science, Wroclaw, Poland) and MDA-MB-231/BO2 (courtesy of Dr. Philippe Clezardin, INSERM U664, France), as well as immortalized normal breast cells (hTERT-HME1 – ME16C, from ATCC) were used. The breast cancer cell lines were cultured in α-MEM medium supplemented with 2 mM L-glutamine (Lonza, Basel, Switzerland), 10% fetal calf serum (FCS; Invitrogen Carlsbad, CA, USA), and antibiotics. The ME16C cell line was cultured in the MEGM Bulletkit medium (Lonza, Basel, Switzerland). All media were supplemented with FBS (Sigma) to a final concentration of 10%. The cells were cultured at 37°C in a 5% CO2.
Immunohistochemistry (IHC)
For the IHC reactions, we used IDC and FBD samples fixed in 10% buffered formalin and embedded in paraffin. To determine the expression of KAT6A and KAT6B, murine monoclonal mouse antibodies directed against KAT6A (1:200; PA5-66742, Thermo Fisher Scientific, Waltham, MA, USA) and KAT6B (1:200; PA5-52251, Thermo Fisher Scientific) were used. IHC was performed using Autostainer Link 48 (DakoCytomation, Glostrup, Denmark) to provide reliable and repeatable conditions.
RNA extraction, cDNA synthesis and real-time PCR reactions
Total RNA from the studied cell lines was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Next, the total RNA was transcribed to cDNA using the High Capacity Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. RT-qPCR was carried out in 20 µL volumes using the iTaq Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, USA) on a 7500 Real-time PCR System. The TaqMan-specific probes used in the experiment (Hs01063026_m1 for KAT6A, Hs00202463_m1 for KAT6B, Hs_01046816_m1 for ESR1, and Hs 99999903_m1 for ACTB as a reference gene) were also obtained from Applied Biosystems. All the above-mentioned reactions were performed in triplicate under the following conditions: activation of polymerase at 50°C for 2 min, initial denaturation at 94°C for 10 min followed by 40 cycles of denaturation at 94°C for 15 s, and annealing and elongation at 60°C for 1 min. The relative mRNA expression of the studied markers was calculated using the ∆∆Ct method.
SDS-PAGE and western blotting
Whole cell lysates from the cell lines were obtained using Cell Lysis Buffer (Thermo Fisher Scientific) with the addition of a cocktail of inhibitors (Sigma, St. Louis MO, USA), 250 units of Benzonase® (Merck Millipore, Bedford, MA, USA), and 2 mM phenylmethanesulfonyl fluoride (PMSF). The lysates were mixed with 4X SDS-PAGE gel loading buffer (200 mM Tris-HCl [pH 6.8], 400 mM DTT, 8% SDS, 0.4% bromophenol blue, and 40% glycerol), loaded on a 10% acrylamide gel, separated by SDS-PAGE under reducing conditions, and then transferred onto a PVDF membrane in the XCell SureLock™ Mini Gel Electrophoresis System (Thermo Fisher Scientific). After protein transfer, the membrane was incubated in a blocking solution (4% BSA in TBST buffer) for 1 h at RT, followed by overnight incubation at 4°C with anti-KAT6A (1:500; PA5-66742, Thermo Fisher Scientific), anti-KAT6B (1:500; PA5-52251, Thermo Fisher Scientific), and anti-ERα (1:750, MA5-14501, Thermo Fisher Scientific). Subsequently, the membrane was rinsed with TBST buffer and incubated for 1 h at RT with secondary donkey anti-mouse antibody conjugated with HRP, diluted 1:3000 (709-035-149; Jackson ImmunoResearch, Mill Valley, CA, USA), and then rinsed and treated with the Immun-Star HRP Chemiluminescent kit (Bio-Rad). Rabbit anti-human β-actin monoclonal antibody (#4970; Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000, was used as the internal control. Western blotting results were analyzed using the ChemiDoc MP System (Bio-Rad).
siRNA transfection
Ambion predesigned siRNAs, including GAPDH siRNA as a positive control and scrambled sequence siRNA as a negative control, were used in the experiments. Specific siRNAs used were as follows: s15534 for KAT6A (MYST-3, Chr.8.) and s108335 for KAT6B (MORF, Chr.10.). MCF-7, an ERα-positive cell line, and MDA-MB-231, an ERα-negative cell line, were grown in 6-well plates as previously described. The concentration of siRNA and quantity of transfection reagent were determined experimentally. Cells were trypsinized, centrifuged at 1000 rpm for 5 min at 4°C, and resuspended in fresh media before transfection. Ambion's siPORT NeoFX (6µl/well) lipid-based transfection reagent and siRNAs (50 nM final concentration) were individually diluted in OptiMEM and mixed. After 10 min, the transfection complexes were overlaid with 2x105 cells/well. Verification of KAT6A and KAT6B silencing and their impact on the ERα expression profile in an estrogen receptor (ER)-positive cell line was conducted.
Statistical analysis
The Shapiro-Wilk test was used to evaluate the normality assumption of the groups examined. The Wilcoxon signed-rank test was used to compare the differences between the LSCC and NMLT groups. Additionally, Spearman’s correlation test was performed to analyze the existing correlations. All statistical analyses were performed using Prism 8.1.0 (GraphPad Software, La Jolla, CA, USA). The results were considered statistically significant at p < 0.05.