Cell lines
Breast cancer cell lines MCF-7, MDA-MB-453, T47D and MDA-MB-231 were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). BT549, HCC1937, and ZR-75-30 cell lines were cultured in Roswell Park Memorial Institute 1640 with 10% FBS. The MDA-MB-361 cell line was cultured in Leibovitz’s L-15 medium with 10% FBS, and the SK-BR-3 cell line was cultured in McCoy’s 5A medium with 10% FBS. All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The SUM159PT cell line was purchased from ProCell (Wuhan, China) and cultured in Ham’s F-12 with 5% FBS, 1 μg/ml of hydrocortisone, 5 μg/ml of insulin, 10 mM HEPES, and 2 mM L-Glutamine. All experiments were performed with mycoplasma-free cells.
Vectors and retroviral infection
Plenti-CMV-puro-P2A-3Flag-spCas9 and Plenti-U6-spg RNA (ALG3)-CMV-EGFP-P2A-blasticidin lentiviruses were purchased from BIiO Technology (Shanghai, China). Endogenous ALG3 knockout was performed according to the manufacturer’s instructions. Briefly, cells were seeded in 24-well plates with a density of 5 × 104 cells per well. When the cells reached ~ 30–40% confluence, they were cultured in basic DMEM containing spCas9 lentiviruses and 5 μg/ml of polybrene for 2 h. Subsequently, completed DMEM containing 10% FBS and 5 μg/ml of polybrene was added and cultured for 12 h. Then, the culture medium was changed with fresh completed DMEM containing 10% FBS. Three days later, puromycin (0.25 μg/ml for SUM159PT and 4 μg/ml for MDA-MB-231) was used to select cells for 5 days. Western blotting was used to confirm whether the selected cells overexpressed spCas9. The selected cells were infected again with lentiviruses containing spgRNA (ALG3), and 3 days later, blasticidin (10 μg/ml for SUM159PT and 15 μg/ml for MDA-MB-231) was used to select cells for 5 days. Western blot was used to identify whether spgRNA (ALG3) was overexpressed in selected cells. After selection, cells were serially diluted in 96-well plates. Human ALG3 complementary DNA (cDNA) were amplified with Real time polymerase chain reaction (RT-PCR) and cloned into the pMSCV-puro-retro vector (Clontech, Beijing, China). Retroviral production and infection were performed, as described previously [38]. Stable cell lines expressing ALG3 were selected for 10 days by treatment with 0.5 μg/ml puromycin for 48 h after infection. Western blot confirmed that stable cell lines were constructed successfully.
Patients and tissue samples
In this study, we used 376 paraffin-embedded breast cancer tissue samples, clinically and pathologically diagnosed at the Sun Yat-Sen University Cancer Center from 2008 to 2012. The clinical and pathological classification and stage were determined according to the 8th edition of the American Joint Committee on Cancer (AJCC) [39]. Histological grade was determined according to the Elston–Ellis modification of the Scarff–Bloom–Richardson system [40]. All 30 patients used to detect expression of ALG family were diagnosed with T2N2M0 breast cancer, who underwent radical mastectomy followed by adjuvant medical therapy and adjuvant radiotherapy delivering 50 Gy in 25 fractions over 5 weeks. And no residual tumor was seen on imagining after the radical mastectomy. As shown in Supplementary Figure S1A, these 30 patients are classified into Luminal (estrogen-receptor and/or progesterone-receptor positive), HER2 (estrogen-receptor and progesterone-receptor negative, HER2 positive), and triple-negative (estrogen-receptor, progesterone-receptor and HER2 negative) subgroups. And p53 status was also summarized in the Supplementary Table S1. The primary evaluation indicator for radiosensitivity were OS and LRFS. All patients provided prior informed consent, and the study was approved by the Institutional Research Ethics Committee. All the tissues were obtained the first time they were diagnosed.
X-ray treatment
Cells in-96 wells plates and mice bearing tumors received X-ray treatment using an RS2000 X-ray Biological Research Irradiator (3 mm copper filter, 160 kV, 25 mA, Rad Source Technologies, GA, USA) at Sun Yat-Sen University Cancer Center. Dosimetry was performed semiannually using an ionization chamber connected to an electrometer system that was directly traceable to a National Institute of Standards and Technology calibration. The mice were narcotized with 60 mg/kg of pentobarbital injected in the abdominal cavity. A 2-mm Cu filter was used for in vivo xenograft experiments.
Real-time RT-PCR
Total RNA samples from the breast cancer cell lines and breast cancer tissues were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracted RNA was pretreated with RNase-free DNase, and ~ 2 μg of RNA from each sample was used for cDNA synthesis primed with random hexamers. For RT-PCR amplification of ALG3 cDNA, an initial amplification step using ALG3-specific primers was performed with activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, primer annealing/extension at 60 °C for 60 s. Next, a final step to melting curve analysis at 95 °C for 10 s, 65 °C for 5 s and 95 °C for 30 s was performed before the reaction mixture was stored at 4 °C. Real-time RT-PCR was used to determine the increase in ALG3 messenger RNA in the primary breast cancer tissue samples. The primers were designed using Primer Express v 2.0 software (Applied Biosystems, Foster City, CA, USA). The forward and reverse primer sequences are shown as Table 1. Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control variability in expression levels and were calculated as 2^[(GAPDHCq) − (ALG3Cq)], where Cq represents the threshold cycle value for each transcript.
Western blotting
Western blotting was performed, as described previously [41], using antibodies against ALG3 (NO. 20290–1-AP, PROTEINTEC, Manchester, UK), TGFBR2(NO. 79424, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), TGFBR1(NO. ab31031, Abcam, Inc., Cambridge science park, UK), phosphorylated SMAD2 (NO. 55041, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), SMAD2 (NO. 5339, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), NANOG (NO. 3580, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), SOX2 (NO. 4900, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), OCT4 (NO.2750, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), CyclinB1 (NO. 4135, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA), CyclinB2 (NO. 21644–1-AP, Proteintech Group, Inc., Rosemont, IL, USA) and CDK4 (NO. 12790, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA). Anti-α-Tubulin mouse monoclonal antibody (NO. 66031–1-Ig, Proteintech Group, Inc., Rosemont, IL, USA) was used to confirm equal loading.
Immunofluorescence staining
Cells were washed by PBS and then fixed with 4% paraformaldehyde for 15 min at 37 °C. After fixation cells were rinsed with PBS, the cells were blocked with 0.1% Triton X-100 containing 1% bovine serum albumin in PBS for 1 h. This was followed by incubation in antibody against TGFBR2 (NO. AF0259, Affinity Biosciences, Cincinnati, USA) and p-smad2 (AF8314, Affinity Biosciences, Cincinnati, USA) for 16 h at 4 °C in a humidified chamber. After washed with PBS, cells were incubated for 1 h at room temperature with fluorescently labeled secondary antibodies. Finally, cells were rinsed in PBS, covered slip with DAPI and examined with a confocal microscope (C1 si, Nikon, Japan).
Caspase-9 or Caspase-3 activity assays
We used the Caspase-9 Colorimetric Assay Kit or Caspase-3 Colorimetric Assay Kit (Keygen, China) to analyze activity of caspase-9 or caspase-3 by spectrophotometry. Firstly, breast cancer cells were suspended in 1 ml ice-cold PBS and washed twice, and then resuspended in lysis buffer. After 30 min incubation on ice, we mixed 50 μl cell suspension, 50 μl reaction buffer, and 5 μl caspase-3/9 substrate, and then incubated it at 37 °C for 1.5 h. The absorbance was measured at 405 nm.
Immunoprecipitation
Cell lyses for protein extraction and co-immunoprecipitation were performed as follows. Cells were lysed in RIPA buffer (150 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl [pH 8.0], 1%Triton X-100 and 0.5% deoxycholate) with complete mini protease inhibitor (Roche). For immunoprecipitation of protein complexes, cell extracts were pre-cleared with Protein G agarose beads (Millipore, 16–266). Then, incubated cell extracts with the polyclone rabbit antibody to TGFBR1 (NO. sc518045, Santa Cruz Biotechnology, Inc., California, USA) or TGFBR2 (NO. ab225902, 1:100, Abcam, Inc., Cambridge science park, UK) for 16 h at 4 °C. Add beads in the cell extracts and incubated for 3 h at 4 °C. Rinsing beads with equilibrium buffer and the protein extraction were then dealt with as for western blot.
Luciferase assay
Cells were seeded in 24-well plates at a density of 2 × 104 cells per well for 24 h. Then, they were transfected with 100 ng of luciferase reporter plasmids or the control-luciferase plasmid, plus 5 ng of pRL-TK Renilla plasmid (Promega, Madison, USA), using the Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer’s instructions. After 24 h, firefly and Renilla signals were measured using the Dual Luciferase Reporter Assay Kit (Promega, Madison, Wisconsin, USA). ARE-luciferase reporter plasmid (Genomeditech, Shanghai, China), HRE-luc (Genomeditech, Shanghai, China), NF-κB-luc (Genomeditech, Shanghai, China), FOXO-luc (Genomeditech, Shanghai, China), SMAD-luc (Genomeditech, Shanghai, China) and TCF-1-luc (Genomeditech, Shanghai, China) were selected to examine activation of antioxidant, hypoxia, NF-κB, PI3K/AKT, TGF-β signaling, and Wnt pathways.
Immunohistochemistry (IHC)
IHC staining and analysis of ALG3 using antibodies against ALG3 (NO. ab151211, Abcam, Inc., Cambridge science park, UK) were performed, as described previously [39, 42]. The ALG3 staining index was analyzed on the basis of the staining intensity (1: no or weak staining; 2: moderate staining; 3: strong staining) and extent (0, 0–25%; 1: 25–50%; 2: 50–75%; 3: 75–100%). A staining score was calculated by multiplying the staining intensity score and the positive cell percentage. The staining intensity and extent values were multiplied and scored as 0, 1, 2, 3, 4, 6, 8 or 9. Using this scoring method, we assessed ALG3 expression in the 376 breast cancer tissue samples. The mean optical density method was used to count inconsistent IHC staining intensities. The best cutoff value was determined using the log-rank test with respect to OS.
Cell counting Kit-8 (CCK-8) assay
Cell viability after radiation was evaluated with Cell Counting Kit-8 (CCK-8; Beyotime, China). MCF-7, ZR-75-30, SUM159PT, and MDA-MB-231 cells were seeded in 96-well plates for 24 h. Then the plates were exposed to 0, 2, 4, 6, (8, 10) Gy X ray. After 72 h, 10 μl of CCK-8 solution was added to each well and incubated for 2 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, California, USA). Cell viability was calculated using the following formula: cell viability = (optical density (OD) value of the treatment group/OD value of the control group) × 100%.
Colony formation assay
Cells were seeded in 6-well plates with at a different density of cells per well and treated with 0, 2, 4, 6 Gy radiation. After 14 days, the cells were then fixed with methanol and the colonies stained with 0.4% crystal violet.
$$ \mathrm{Surviving}\ \mathrm{Fraction}=\mathrm{Colonies}/\left(\mathrm{Input}\ \mathrm{cells}\times \mathrm{Plating}\ \mathrm{Efficiency}\right) $$
$$ \mathrm{Plating}\ \mathrm{Efficiency}=\mathrm{Colonies}\ \mathrm{in}\ \mathrm{control}\ \mathrm{group}/\mathrm{Input}\ \mathrm{cells}\ \mathrm{in}\ \mathrm{control}\ \mathrm{group}\times 100\% $$
The mean surviving fraction and its standard error were calculated from 3 independent experiment for each cell line. Survival curves were fitted using GraphPad Program by nonlinear regression analysis based on multi-target single-hit model. Clone survival fraction (S) with increasing dose (D) can be described using S = 1 – (1 – e−D/D0) N = 1-(1-e-kD)N. Based on the results of the nonlinear regression analysis, we got k and N values. D0 = 1/k, and Dq = D0 × ln (N). N value represents the comprehensive degree of sub-damage repair capacity and the sensitivity to radiation damage. D0 value represents the sensitivity to radiation damage. The smaller the D0 value, the smaller the dose is required to kill a certain proportion of cells; Dq reflects the ability against DNA damage. The greater the Dq value, the greater the dose is required to induce cells death.
Tumor sphere formation assays
Five hundred cells were seeded in 6-well ultra-low attachment plates and cultured in suspension in serum-free DMEM: F12 medium (BioWhittaker) supplemented with 0.4% BSA, 2% B27 (Invitrogen), 20 ng/ml of epidermal growth factor, 20 ng/ml of basic fibroblast growth factor, and 5 μg/ml of insulin (PeproTech, Rocky Hill, NJ, USA) for 10 days. The number of spheres formed (tight, spherical, nonadherent masses > 50 μm in diameter) was counted, and images were captured under an inverted microscope (C1 si, Nikon, Japan).
$$ \mathrm{Sphere}\ \mathrm{formation}\ \mathrm{efficiency}\ \left(\%\right)=\left(\mathrm{Colonies}/\mathrm{Input}\ \mathrm{cells}\right)\times 100 $$
Flow cytometry assay
Cells were dissociated with trypsin, resuspended at 1 × 106 cells/ml in DMEM containing 2% FBS, and then incubated at 37 °C for 30 min with or without 100 μM verapamil (Sigma-Aldrich, St. Louis, MO, USA) to inhibit adenosine triphosphate-binding cassette transporters. The cells were subsequently incubated with 5 μg/ml Hoechst 33342 (Sigma-Aldrich) at 37 °C for 90 min or stained at 4 °C with purified CD44 (ab157107, rabbit immunoglobulin G [IgG]; Abcam)–fluorescein isothiocyanate (FITC) (ab6717, rabbit IgG; Abcam), CD24 (ab31622, mouse IgG1; Abcam)-PE-CY7 (ab130790, mouse IgG2b; Abcam), or annexin V-FITC–propidium iodide (PI) (KGA106). Finally, the cells were incubated on ice for 10 min and washed with ice-cold phosphate-buffered saline prior to flow cytometry analysis. Samples were analyzed and sorted on Beckman–Coulter MoFlo and Beckman–Coulter gallios, respectively, with data analyzed using FlowJo software (Tree Star Inc., USA).
Xenograft tumor model
A xenograft tumor model was constructed, as described previously [43]. Female BALB/c nude mice (4–5 weeks old, 18–20 g in weight) were purchased from Slac-Jingda Laboratory Animal (Hunan, China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. The mice’s flanks were injected subcutaneously with 5 × 106 MCF-7, ZR75–30, MDA-MB-231 and SUM159PT cells. The tumors were examined every week, including length and width, using calipers, and tumor volumes were calculated. After 14 days, the mice from ALG3-transduced, ALG3 vector, ALG3 knocked-out, and control groups underwent six fractions with 2 Gy/fraction radiation. Next, we compared the speed of tumor growth between groups with different ALG3 expression. On day 63, the mice were euthanized and the tumors excised and measured.
Statistical analysis
Statistical analyses were performed using SPSS v22 statistical software (SPSS Inc., Chicago, IL, USA). Comparison between categorical variables was performed by chi-square test. Pearson or Spearman correlation test was used to examine correlations between two continuous variables when indicated. Survival curves were plotted using the Kaplan–Meier method and the log-rank test, and survival data were evaluated using multivariate cox regression analysis. P < 0.05 was considered statistically significant. Two-way ANOVA with Bonferroni’s post test, one-way ANOVA with Tukey’s multiple comparison test, and Student’s t test were used to evaluate the difference between the experimental group and the control group.