Hexokinase activity assay

Hexokinase activity in whole-cell lysates and mitochondrial fractions was measured using a standard glucose-6-phosphate (G6P) dehydrogenase (G6PDH)-coupled spectrophotometric assay as described previously⁵⁶. Whole-cell lysates were prepared by brief sonication in homogenization buffer containing 45 mM Tris-HCl, 50 mM KH2PO4, 10 mM glucose, and 0.5 mM EGTA. Hexokinase activity was measured as the total glucose-phosphorylating capacity of whole-cell lysates in a final assay mixture containing 50 mM triethanolamine chloride, 7.5 mM MgCl2, 0.5 mM EGTA, 11 mM monothioglycerol, 4 mM glucose, 6.6 mM ATP, 0.5 mg/ml NADP, and 0.5 U/ml G6PDH, pH 8.5. Hexokinase activity in each sample was calculated as the coupled rate of NADPH formation by the Lambert–Beer law as follows: [(A340/t)] × dilution factor/[protein], where (6.22 mM⁻¹cm⁻¹) is the extinction coefficient for NADPH at 340 nm (t = time, [protein] = protein concentration).

An enzymatic fluorometric assay to quantify G6P was performed following the method described previously⁵⁹ with minor modifications. Briefly, samples were extracted from 5 × 10⁶ cells with MeOH/CHCl₃ and stored at −80 °C. Just prior to the assay, the extracted samples were dissolved in 50 μl of Millipore water, and then 10 μl of each extraction sample were incubated for 30 min at room temperature in a 96-well plate with 90 μl of an assay cocktail containing 50 mM triethanolamine (TEA, pH 7.6), 1.0 mM MgCl2, 100 μM NADP+, 10 μM resazurin, 1.5 U/ml G6PD, and 0.2 U/ml diaphorase). In this assay, G6P is oxidized by G6P dehydrogenase in the presence of NADP+, and stoichiometrically generated NADPH is then amplified by the diaphorase–resazurin system. Fluorescence at 590 nm was measured using excitation at 530 nm. Background fluorescence was corrected by subtracting the value of the blank for each sample, and G6P concentrations were calculated from a standard curve. Fluorescence was measured using a Tecan Infinite M200 PRO plate reader in 96-well black assay plates.

For measuring MCL1 protein stability in MI5-4 CHO cells and in MI5-4 CHO cells expressing HK2 or for measuring endogenous SNAIL protein stability in 4T1 and 4T1shHK2 cells, cells were plated on 6-well plates at a seeding density of 2 × 10⁵ cells/well. Cells were treated with cycloheximide (100 μM) for the indicated time points. For wild-type GFP-tagged or 6SA GFP-tagged SNAIL, 1.5 μg of plasmid was transiently transfected by using Lipofectamine 2000 overnight.

Enzyme-linked immunosorbent assays were conducted in Pierce Nickel (His) or Glutathione (GST) 96-well coated plates. The plates were respectively coated with His-PRKAR1a (50 nM; Sino Biological) or GST-GSK3β (50 nM; Abcam), in coating buffer (phosphate-buffered saline containing 0.5 mM phenylmethanesulfonyl fluoride and 1 mM dithiothreitol) and incubated overnight at 4 °C. Thereafter, unbound protein was removed by washing the wells three times with 100 μl of washing buffer (phosphate-buffered saline containing 0.05% Tween 20) and blocking buffer (coating buffer containing 0.6% skimmed milk powder and 0.05% Tween 20) was added (100 μl, 1 h, room temperature). After removal, monitoring of interactions of PRKAR1a or GSK3β with HK2 was carried out in coating buffer and increasing concentration of Myc-HK2 protein (OriGene; 0–64 nM, 40 μl/well, 2 h, room temperature). For G6P-induced binding inhibition, 30 min before the end of the incubation, G6P (0–100 μM) is added directly to the wells. Unbound protein was removed by washing the wells three times with 100 μl of washing buffer and bound Myc-HK2 was detected with monoclonal anti-Myc HRP-conjugated antibody (Abcam; 1:5000 in blocking buffer, 1 h, room temperature). Bound PRKAR1a or GSK3β were detected by incubation with rabbit anti- PRKAR1a antibody (Cell Signaling; 1:2000 in blocking buffer) or mouse anti-GSK3β antibody (Millipore; 1:2000 in blocking buffer) and HRP-conjugated anti-rabbit or mouse IgG (1:3000, 1 h, room temperature). For each assay, non-specific binding is also conducted as described in figures.

Each antibody incubation step was followed by washing. The HRP reaction was initiated by addition of 3,3′,5,5′-tetramethylbenzidine enzyme-linked immunosorbent assay substrate solution (Sigma) and terminated after 30 min by adding H2SO4 (2 M; 50 μl/well). The colored reaction product was quantified by measuring A450 nm in a Cytation 1 plate reader (Biotek).

Binding assay is conducted on Pierce Nickel-coated 96-well plates and after successive incubation with His-PRKAR1a and Myc-HK2 as described above, unbound proteins will be washed and blocking buffer will be added for 1 h at room temperature. Thereafter, GST-GSK3β is added at increasing concentration (0–64 nM; 40 μl/well, 2 h, room temperature). For G6P-induced binding inhibition, 30 min before the end of the incubation, G6P (0–1000 μM) is added directly to the wells. Unbound protein was removed by washing the wells as described above and bound GST-GSK3β was detected with monoclonal anti-GST HRP-conjugated antibody (Abcam; 1:5000 in blocking buffer, 1 h, room temperature). Bound PRKAR1a or HK2 were detected by incubation with rabbit anti- PRKAR1a antibody (Cell Signaling; 1:2000 in blocking buffer) or rabbit anti-HK2 antibody (Cell Signaling; 1:2000 in blocking buffer) and HRP-conjugated anti-rabbit IgG (1:3000, 1 h, room temperature). For each assay, non-specific binding is also conducted as described in figures and HRP reaction is conducted as described above.

Enzyme-linked immunosorbent assays were conducted in Pierce Nickel-8-well-strip-coated plates. The plates were coated with His-HK2 (50 nM; Creative Biomart) in coating buffer and incubated overnight at 4 °C. Unbound proteins is then washed and blocking buffer is added for 1 h at room temperature. Thereafter, Myc-PRKAR1α (32 nM; Creative Biomart), PKAc (20 nM; Promega) and GST-GSK3β (32 nM; Abcam) successively, each for 1 h incubation at room temperature, and unbound protein will be carefully washed after each incubation. After all proteins are bound, kinase buffer (1 mM ATP and 10 mM MgCl₂ in Tris 20 mM, pH 7.4) with or without cyclic AMP (10 µM) is added to all appropriate wells (1 h; room temperature). After incubation, kinase buffer is washed and appropriate antibody will be added to the wells de detect bound proteins (P-GSK3β, total GSK3, PRKAR1α, HK2, and PKAc) and incubated overnight at 4 °C. After washing, total bound GST-GSK3β was detected with monoclonal anti-GST HRP-conjugated antibody, and bound P-GSK3β, PRKAR1α, HK2 and PKAc antibodies are detected with HRP-conjugated anti-rabbit IgG (1:3000, 2 h, room temperature). The HRP reaction is conducted as described above.

For G6P-induced binding inhibition, G6P (1 mM) is added 30 min before the end of the incubation with GST-GSK3β, before the addition of kinase buffer.

Negative control reactions are conducted as described above, in absence of His-HK2 or Myc-PKAR1α.

Binding assay is conducted on Pierce Nickel-coated 8-well strip plates and after successive incubation with His-PRKAR1α and Myc-HK2 as described above, unbound proteins will be washed and blocking buffer will be added for 1 h at room temperature. Thereafter, FMP-API-1 is added directly to the wells 30 min before the end of the incubation with Myc-HK2 at increasing concentration (0–1000 μM). Unbound protein was removed by washing the wells as described above and bound Myc-HK2 was detected with monoclonal anti-Myc HRP-conjugated antibody (Abcam; 1:5000 in blocking buffer, 1 h, room temperature). HRP reaction is then conducted as described above.

For metabolic tracing in A549 cells, 7.0 × 10⁵ cells were plated onto 6-well plates for next day pulse-labeling with 25 mM [1,2-¹³C] glucose (Cambridge Isotope Laboratories). Isotope labeling experiments were performed for 4 h, and extra plates were included for cell counts. At the time of collection, plates were washed twice with 1 ml of (9 g/l) NaCl. Then, cells were incubated with 600 μl mixed solvent (water:methanol:acetonitrile = 1:1:1) containing 2 μl of 2 mg/ml Norvaline (SigmaAldrich) dissolved in distilled water as an internal standard, and then scraped down with cell scrapers. The solution was shaken at 1200 rpm for 30 min and centrifuged at 16,000 × g for 15 min at 4 °C. The supernatant (280 μl) was transferred to a clean tube and lyophilized under nitrogen gas. The lyophilized samples were derivatized with 15 µl of 2 wt% methoxylamine hydrochloride (Thermo Fisher) for 60 min at 42 °C. Next, 35 µl of N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) +1% tert-butyldimetheylchlorosilane (t-BDMCS) (SigmaAldrich) was added and the samples were incubated for 60 min at 75 °C. The derivatized samples were then centrifuged at 16,000 × g for 5 min at 4 °C, and the supernatant (1 μl) was subjected to GC-MS measurement.

GC-MS analysis was performed using an Agilent 7890B GC equipped with a DB-5ms column (30 m × 0.25-mm inner diameter; film thickness, 0.25 μm; Agilent J&W Scientific). The front inlet temperature was 280 °C and helium flow was maintained at 1.428 ml/min. For intracellular lactate analysis, the column temperature was held at 60 °C for 1 min followed by 1-min of run time, rising at 10 °C/min to 320 °C and holding for 1 min followed by a post run of 320 °C for 9 min. For the analysis of G6P and 6PG, the initial rate was held at 60 °C for 1 min followed by 1-min of run time, rising at 10 °C/min to 325 °C and holding for 10 min followed by a post run of 60 °C for 1 min^60,61. The peak area of each quantified ion was calculated and corrected for natural isotope abundances by following the procedure by Fernandez et al.⁶², and then normalized by the peak area of norvaline as an internal standard and cell number.

For metabolic tracing, in 4T1 and 4T1shHK2 cells, the cells were plated on 6 cm plate in triplicate (3 × 10⁵) in 5 mM glucose DMEM media for 48 h. Cells were washed in PBS and 5 mM of uniformly labeled C-13 glucose in DMEM with 10% dialyzed FBS was added for 5 min following extraction in 1 ml of cold 90/10 acetonitrile/water. Cells were collected using a cell scrapper, vortexed for 2 min, and centrifuged at highest speed for 5 min. The supernatant was transferred to clean Eppendorf tube and sent to MD Anderson Cancer Center’s Proteomics and Metabolomics Core Facility for analysis by Ion chromatography mass spectrometry (IC-MS). One 6 cm plate was collected for western blot. Cell number was counted on extra 6-well plate and protein concentration were quantified with remaining cell pellet.

Extracellular lactate concentrations were measured using a YSI 2700 Bioanalyzer (YSI).

C57BL/6 MMTV-PyMT and HK2^f/f;UBC-Cre^ERT2 mice were previously described⁵¹. MMTV-PyMT;HK2^f/f mice were crossed with HK2^f/f;UBC-Cre^ERT2 or HK2^f/f mice to generate experimental and control mice on a C57BL/6 background. Once a primary tumor was palpable (~12 weeks old), 0.1 ml of 30 mg/ml tamoxifen was injected IP for 7 consecutive days to systemically delete HK2 as previously described⁵¹. At the tumor endpoint (20% weight loss from baseline, 15% weight gain compared to aged-matched controls, tumor size greater than 15% of the body weight, tumor mass >2 cm, tumor ulceration, pallor, respiratory distress, or inability to ambulate), the mice were euthanized, and their lungs were analyzed for metastases.

MMTV-rtTA, Tet(O)Cre, and LSL-luc mice were purchased from Jackson Laboratory.

MMTV-PyMT;Hk2^f/f;LSL.Luc;MMTV.rtTATet(O)Cre mice were generated by first crossing MMTV-rtTA mice with LSL.Luc and Tet(O)Cre mice to generate LSL.Luc;MMTV.rtTATet(O)Cre mice. These mice were crossed with MMTV-PyMT;Hk2^f/f mice.

Balb/cJ mice were purchased from Jackson Laboratory and NOD.Cg-Prkdcscid (NOD-F) mice from TACONIC.

All animal work was conducted according to the ethical regulations set forth by the federal government, as reviewed and approved through the Office of Animal Care and Institutional Biosafety (OACIB) within the Office of the Vice Chancellor for Research at the University of Illinois, Chicago.

Mammary tumor tissues and PBS-inflated lung lobes were collected, and macroscopic lung lesions were counted visually. The tissues were fixed in 10% formalin for 48 h. Tissues were paraffin embedded and sectioned for hematoxylin and eosin (H&E) staining (5-µm sections). Microscopic lung lesions were counted using a microscope.