Mice
Six- to 8-week-old female BALB/c athymic nude mice (Hsd:Athymic Nude-Foxn1nu) were purchased from Envigo (Italy) and used to generate a xenograft mouse BC model (see the “Tumor models” section). Mice were housed in temperature- and light-controlled conditions (12-h light cycle) with food and water ad libitum. Mice were acclimatized for 2 weeks after their arrival. 129SvJ female mice heterozygous for Sirt6 (Sirt6+/−) were a kind gift from Dr. Raul Mostoslavsky (MGH Cancer Center, Boston, MA, USA) [18]; they were used to generate a transgenic model of spontaneous BC (see the “Tumor models” section). Male mice heterozygous for MMTV-PyMT (MMTV-PyMT+/−) in a 129/Ola genetic background were provided by Dr. Thorsten Berger (The Campbell Family Institute for Breast Cancer Research Ontario Cancer Institute) [19]. The MMTV-PyMT colony was maintained through heterozygous males because the heterozygous females develop tumors early and are therefore unable to breastfeed any puppies. Three- to 9-month-old Sirt6+/− female mice were bred with MMTV-PyMT+/− male mice to generate two different groups of interest: MMTV-PyMT+/−; Sirt6+/+ control mice and MMTV-PyMT+/−; Sirt6+/− experimental mice. The mammary glands were isolated from 12-week-old Sirt6+/+ and Sirt6+/− mice by standard procedures, flash-frozen, and stored at − 80 °C until utilization for RNA isolation, ATP, and AMP measurements and respiratory complex activity assays. All animal experiments were performed in accordance with the relevant guidelines and regulations (Italian 26/2014 and EU 2010/63/UE directives) and were approved by the Licensing and Ethical Committee (OPBA) of Ospedale Policlinico San Martino IRCCS and by the Italian Ministry of Health.
Cell lines
MCF7, MDA-MB-231, and Phoenix cell lines were purchased from ATCC and were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin (50 U/mL) (LifeTechnologies, Italy).
Viral production and transduction
The retroviral plasmids for silencing SIRT6 (short hairpin#2 - sh2) and for overexpressing wild-type (WT) or catalytically inactive (H133Y) SIRT6, and the respective vectors (PRS for the shRNA and pBABEpuro for the expression plasmids), were described previously [20, 21]. For retroviral transductions, 1 × 106 Phoenix cells were plated in 4 ml medium in 6-cm dishes and allowed to adhere for 24 h. Thereafter, cells were transfected with 4 μg of plasmid DNA using TransIT-293 (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. Viral supernatants were harvested after 36, 48, 60, and 72 h and used to infect MCF7 cells (5 × 105) and MDA-MB-231 (3 × 105) cells in 10-cm dishes in the presence of 5 μg/ml protamine sulfate. Successfully infected cells were selected using 1.5 μg/ml puromycin (MCF7) or 1 μg/ml puromycin (MDA-MB-231).
Western blot analysis
After transduction, 6 × 105 MCF7 and 4 × 105 MDA-MB-231 cells were plated in 10-cm dishes and allowed to adhere for 48 h. Thereafter, cells were lysed in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, Protease Inhibitor Cocktail, and Phosphatase Inhibitor Cocktail 2 from Sigma Aldrich], and the protein concentration was determined according to a standard Bradford assay. Proteins (35 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore, Vimodrone, Italy). Proteins of interest were detected with the following antibodies: anti-SIRT6 (1:1000, #2590 Cell Signaling), anti-Phospho-AMPK (1:1000, PA5-17831 Thermo Scientific), anti-AMPK (1:1000, PA5-29679 Thermo Scientific), anti-pyruvate dehydrogenase (1:1000, #3205 Cell Signaling), anti-GAPDH (1:1000, #5174 Cell Signaling), anti-Vinculin H-300 (1:200, sc-5573 Santa Cruz Biotechnology), anti-SIRT6 (1:1000, LS-C49020 LifeSpan BioSciences; for detecting mouse Sirt6), and anti-αtubulin (1:1000, DM1A: sc-32293, Santa Cruz Biotechnology). Band intensities were quantified by the Quantity One SW software (Bio-Rad Laboratories, Inc.) using standard ECL.
Tumor masses were excised from xenograft mice and homogenized in ice-cold lysis buffer with an electric homogenizer. The samples were maintained in constant agitation for 2 h at 4 °C, on an orbital shaker in a cold room. Finally, the samples were centrifuged for 20 min at 12,000 rpm at 4 °C in a microcentrifuge, the supernatants were collected, and the protein content was evaluated by Bradford assay.
Quantitative real-time PCR
RNA extraction, cDNA synthesis, and quantitative real-time polymerase chain reaction (QPCR) were performed as described elsewhere [22]. Gene-specific primers were purchased from Sigma-Aldrich (Italy) or Thermo Fisher (Italy) and are listed in Supplementary Table 1. Comparisons in the gene expression were performed using the 2−ΔΔCt method. β-Actin was used as the housekeeping gene.
Mitochondrial mass determination
For mitochondrial mass determination, 2 × 105 MDA-MB-231 cells were stained with 200 nM Mitotracker deep red (M22426, Thermo Fisher, Italy) according to the manufacturer’s instructions and immediately analyzed by flow cytometry (FACS Aria, Becton Dickinson, Germany) by acquiring 10,000 events. The Mitotracker deep red mean fluorescence intensity (MFI) was also estimated. Changes in mitochondrial mass as a consequence of either SIRT6 overexpression or silencing were also evaluated by estimating mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratio. To this end, 106 MDA-MB-231 cells were washed with PBS, centrifuged at 1.200 rpm for 5 min at 4 °C and resuspended in 100 μl lysis buffer (100 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM EDTA, 0.2% SDS) with freshly added proteinase K (0.2 mg/ml). After 2 h incubation at 56 °C, samples were processed as described in "DNA extraction and mouse genotyping" (see below). One hundred nanograms of DNA was used to perform QPCR with SYBR green-based detection. Relative mtDNA:nDNA ratio was calculated using the 2−ΔΔCt method by targeting the nuclear-encoded gene, human B2M, and the mitochondrial-encoded gene, human tRNALeu. Gene-specific primers are listed in Supplementary Table 1.
Analysis of mitochondrial morphology by Mitotracker deep red
The analysis of mitochondrial morphological features was performed as described elsewhere [23]. In brief, 4 × 104 MDA-MB-231 cells were plated on glass coverslips (Thermo Scientific Nunc Lab-Tek II chamber slide system, Italy) and allowed to adhere overnight. Thereafter, cells were stained with Mitotracker deep red (Thermo Fisher, Italy) according to the manufacturer’s instructions and analyzed by confocal microscopy. Images were collected using a three-channel TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Mitochondrial solidity was defined as the fraction of pixels contained with a convex polygon (fitted around a mitochondrion) that is also mitochondrial pixels. Low solidity (close to 0.0) tends to describe highly tortuous mitochondria that are not uniform in shape, while high solidity values (close to 1.0) tend to describe mitochondria that are more uniform in shape and do not contain a high level of branching. Mitochondrial perimeter was defined as the number of exterior mitochondrial pixels multiplied by the length of the pixels, in microns. Circularity was calculated by the following formula: circularity = 4π × (area/perimeter2). Mitochondria exhibiting a perfect circular shape have a circularity value close to 1.0, whereas more elongated mitochondria have a circularity value that is close to 0.0.
Tumor models
To generate the MDA-MB-231 BC xenograft model, 2 × 106 MDA-MB-231 cells engineered with the PRS vector (VECTOR) or with a SIRT6-targeting shRNA (SIRT6-sh) were subcutaneously injected into both flanks of female BALB/c athymic nude mice. Tumor growth was monitored over time measuring tumor sizes with a manual caliper, and the tumor volume was registered twice a week. Mice were sacrificed when the tumor reached a volume of about 1.5 cm3 [the maximal tumor volume permitted by our Institutional Animal Care and Use Committee (IACUC)]. A genetically modified model that spontaneously develops breast tumors was also used (see above). MMTV-PyMT+/−; Sirt6+/+ and MMTV-PyMT+/−; Sirt6+/− mice were monitored palpating the mammary glands: when the tumors appeared as palpable masses, the age of the mice was recorded to study tumor latency and mouse survival. Tumor volume was calculated using the following formula: tumor volume = (w2 × W) × π/6, where w and W are “minor side” and “major side” (in mm), respectively. Blood glucose was measured in blood samples that were obtained from the tail of 3-month-old Sirt6+/+, Sirt6+/−, MMTV-PyMT+/−; Sirt6+/+, and MMTV-PyMT+/−; Sirt6+/− mice using Glucomen Areo 2k (Menarini, Italy). Organs [heart, lung, kidney, spleen, and mesenteric visceral adipose tissue (VAT)] from 4-week-old MMTV-PyMT+/−; Sirt6+/+ and MMTV-PyMT+/−; Sirt6+/− mice were collected by standard procedures, flash-frozen, and stored at − 80 °C until utilization for RNA isolation.
DNA extraction and mouse genotyping
DNA extraction from mouse tails was performed according to the following protocol: approximately 4 mm mouse tail was digested with 500 μl tail lysis buffer [5 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), 200 mM NaCl, in 100 mM Tris-HCl, pH 8.0] containing 200 μg/ml proteinase K, in a 1.5-ml tube at 56 °C overnight with agitation (1,000 rpm on a heated shaker). The day after, samples were centrifuged at 14,000 rpm for 10 min at room temperature for debris removal. The supernatant was transferred to a clean tube with 500 μl isopropanol and inverted until DNA precipitation was observed. Subsequently, samples were centrifuged at 14,000 rpm for 5 min at RT, the supernatant was discarded, and the DNA pellet was washed by adding 300 μl of 70% ethanol and centrifuged at 14,000 rpm for 5 min at RT. After centrifugation, the ethanol solution was removed, and the DNA pellet was dried at RT or in a desiccator. Finally, the DNA pellet was resuspended in 35–100 μl DNase-free water. Genotyping was performed using Multiplex PCR Master Mix 2x (BR0200801, Biotechrabbit, Germany) to determine the presence of WT or KO allele of Sirt6 and of PyMT genes. For Sirt6 gene, three oligonucleotides were used, two specific for WT or KO allele and one in common for the two different alleles (WT: TTTCGTATAAGTCCAAGCCC, KO: GCAATAGCATCACAAATTTCAC, COMMON: GGAAGGACCTGGACAAG). WT allele primer pair (WT and COMMON) amplifies a fragment of 422 bp spanning across exon 8, while KO allele primer pair (KO and COMMON) amplifies a fragment of 300 bp spanning across exon 8. For PyMT gene, two oligonucleotides were used (PyMT 1: GGAAGCAAGTACTTCACAAGG and PyMT 2: GGAAAGTCACTAGGAGCAGGG) to amplify a fragment of 600 bp. PCR products were analyzed by gel electrophoresis with 2% precast agarose gels (54813, 2% seakem gold agarose, Lonza, Italy) at 100 V. The amplification products were visualized with a ChemiDoc XRS (BioRad, Italy) instrument.
Mammary tissue morphologic analysis
To prepare whole mounts, the fourth inguinal mammary gland was excised from 30-day-old female MMTV-PyMT+/−; Sirt6+/+ and MMTV-PyMT+/−; Sirt6+/− mice and spread out on a pre-cleaned microscope slide. The gland was fixed in Carnoy’s solution (75% ethanol–25% glacial acetic acid) overnight at RT. The day after, the slides were hydrated in 70% ethanol and water and stained overnight with carmine alum (07070, STEMCELL Technologies, Italy). After staining, the slides were rinsed in water, dehydrated in increasing concentrations of ethanol, and cleared in histolemon (454915, Carlo Erba, Italy). Photographic images of the whole mounts were acquired by Nikon SMZ1270 microscope using the X-Entry software. Terminal end buds (TEB) were counted in the whole mammary gland.
Histopathological and immunohistochemical analysis of mammary tumors
After being sacrificed by cervical dislocation, MMTV-PyMT+/−; Sirt6+/+ and MMTV-PyMT+/−; Sirt6+/− mice were refrigerated and immediately sent to the pathological anatomy laboratory. Each animal was fixed by immersion in 10% buffered formalin for 12–18 h, after inoculation of the fixative intraperitoneally and intrathoracically. Subsequently, each animal was eviscerated en-block and the organs seriated with consecutive sections in the craniocaudal sense. Bilateral paramedian incisions along the breast lines were also performed to sample mammary tumors. All samples were routinely processed and paraffin-embedded to obtain 3-μm-thick histological sections stained with hematoxylin-eosin. Microvessel density was determined by CD34 staining (anti-CD34, clone QBEnd/10, Ventana) as described in [24].
Cell homogenate preparation for ATP and AMP measurements and respiratory complex activity assays
To remove the growth medium, cells were centrifuged at 1000g for 2 min. The pellet was suspended in phosphate-buffered saline (PBS) buffer and sonicated on ice, 2 times for 10 s, with a 30-s break. The total protein concentration was estimated by the Bradford method [25].
Evaluation of intracellular ATP and AMP content
Quantification of ATP and AMP was based on the enzyme coupling method [26]. Twenty micrograms of total proteins was used. Briefly, ATP was assayed spectrophotometrically at 340 nm, following NADP+ reduction, at 25 °C. The reaction mixture contained the following: 1 mM NADP+, 10 mM MgCl2, 5 mM glucose, and 100 mM Tris-HCl, pH 7.4, in 1 ml final volume. Samples were analyzed before and after the addition of purified hexokinase and glucose-6-phosphate dehydrogenase (4 μg; HK+G6PD, HKG6PDH-RO, Sigma-Aldrich, Italy). AMP was assayed spectrophotometrically at 340 nm, following NADH oxidation. The reaction mixture contained the following: 75 mM KCl, 5 mM MgCl2, 0.2 mM ATP, 0.5 mM phosphoenolpyruvate, 0.2 mM NADH, 10 IU adenylate kinase (AK, M3003, Sigma-Aldrich, Italy), 25 IU pyruvate kinase plus 15 IU lactate dehydrogenase (PK+LDH, Sigma-Aldrich, Italy), and 100 mM Tris-HCl pH 8.0.
Oxymetric analysis
Oxygen consumption was measured with an amperometric oxygen electrode (Unisense) in a closed chamber, magnetically stirred, at 37 °C. For each assay, 2 × 105 cells were used. After cell permeabilization with 0.03 mg/ml digitonin for 10 min, samples were suspended in 137 mM NaCl, 5 mM KH2PO4, 5 mM KCl, 0.5 mM EDTA, 3 mM MgCl2, and 25 mM Tris-HCl, pH 7.4. To activate the pathway composed of complexes I, III, and IV, 5 mM pyruvate and 2.5 mM malate were added. To activate the pathway composed of complexes II, III, and IV, 20 mM succinate was used [27].
Evaluation of F1Fo-ATP synthase activity
F1Fo-ATP synthase (ATP synthase) activity was detected by measuring the ATP production by the highly sensitive luciferin/luciferase method. The assays were conducted at 37 °C, for 2 min, and data were collected every 30 s. Cells (1 × 105) were added to the incubation medium (0.1 ml final volume), which contained 50 mM KCl, 1 mM EGTA, 2 mM EDTA, 5 mM KH2PO4, 2 mM MgCl2, 0.6 mM ouabain, 1 mM P1,P5-Di(adenosine-5′) pentaphosphate, 0.040 mg/ml ampicillin, 0.2 mM adenosine diphosphate (ADP), 10 mM Tris-HCl pH 7.4, and the metabolic substrates (5 mM pyruvate + 2.5 mM malate or 20 mM succinate). Cells were equilibrated for 10 min at 37 °C, and then ATP synthesis was induced by the addition of 0.2 mM ADP. ATP synthesis was measured using the luciferin/luciferase ATP bioluminescence assay kit CLSII (11699695001, Roche) and a Luminometer (GloMax® 20/20 Luminometer, Promega). ATP standard solutions in the concentration range 10−10–10−7 M were used for calibration [27].
Respiratory complex activity assay
The activity of the redox complexes I, III, and IV was measured, in a double beam spectrophotometer (UNICAM UV2, Analytical S.n.c.), at 25 °C. For each assay, 50 μg total proteins were employed, and the reaction was followed for 5 min, collecting data every 1 min [28]. The enzymatic activity was expressed as mIU/mg total protein (nanomoles/min/mg protein). Complex I (NADH-ubiquinone oxidoreductase) was assayed following the reduction of ferrocyanide, in the presence of NADH, at 420 nm; the reaction mixture contained 30 mM NADH, 40 mM potassium ferrocyanide, and 40 μM antimycin A in 10 mM phosphate buffer pH 7.2. Complex III (cytochrome c reductase) activity was measured at 550 nm following the reduction of oxidized cytochrome c. The reaction mixture contained 10 mM phosphate buffer pH 7.2, 0.03% oxidized cytochrome c, and 0.5 mM KCN. Complex IV (cytochrome c oxidase) was assayed following the oxidation of ascorbate-reduced cytochrome c at 550 nm, in a solution containing 10 mM phosphate buffer pH 7.2, 0.03% reduced cytochrome c, and 40 μM antimycin A. In both assays, the cytochrome c extinction coefficient was considered 19.1 × 10−3 M−1 cm−1, at 550 nm.
Evaluation of pyruvate dehydrogenase expression and activity
The expression and activity of pyruvate dehydrogenase (PDH) were performed using the “Pyruvate dehydrogenase (PDH) Combo (Activity + Profiling) Microplate Assay Kit” provided by Abcam (ab110671, Abcam), following the manufacturer’s instructions. For each sample, 4 × 106 MDA-MB-231 cells were used.
Quantification and statistical analysis
Statistical analyses were performed with the GraphPad Prism software version 5 (GraphPad Software, San Diego, USA). Data are shown as mean ± SD. All parameters were tested by two-tailed Student’s t test (in all in vitro experiments), by two-way ANOVA followed by Bonferroni post hoc test (in vivo xenografts experiments), or log rank test (in vivo survival and latency experiments). P values less than 0.05 were considered significant.