The interferon-inducible protein viperin controls cancer metabolic reprogramming to enhance cancer progression

Metabolic reprogramming is an important cancer hallmark. However, the mechanisms driving metabolic phenotypes of cancer cells are unclear. Here, we show that the interferon-inducible (IFN-inducible) protein viperin drove metabolic alteration in cancer cells. Viperin expression was observed in various types of cancer and was inversely correlated with the survival rates of patients with gastric, lung, breast, renal, pancreatic, or brain cancer. By generating viperin knockdown or stably expressing cancer cells, we showed that viperin, but not a mutant lacking its iron-sulfur cluster–binding motif, increased lipogenesis and glycolysis via inhibition of fatty acid β-oxidation in cancer cells. In the tumor microenvironment, deficiency of fatty acids and oxygen as well as production of IFNs upregulated viperin expression via the PI3K/AKT/mTOR/HIF-1α and JAK/STAT pathways. Moreover, viperin was primarily expressed in cancer stem-like cells (CSCs) and functioned to promote metabolic reprogramming and enhance CSC properties, thereby facilitating tumor growth in xenograft mouse models. Collectively, our data indicate that viperin-mediated metabolic alteration drives the metabolic phenotype and progression of cancer.

pΔVPR plasmid and the pVSVG plasmid were also used. HEK-293T cells were co-transfected with three plasmids, 12 μg of the lentiviral plasmid, 12 μg of the pΔVPR plasmid and 12 μg of the pVSVG plasmid, by using lipofectamine 2000 (Thermo Fisher Scientific 11668-019), and incubated at 37ºC for 16-24 h. The co-transfected cells were shifted to 32ºC. After 1 day, supernatants containing lentiviruses were collected every 16-24 h three times. Cancer cell lines (MKN1, MKN28, and AGS) were spin infected with the supernatants containing viruses for 90 min (1,455 × g) at 32ºC in the presence of 8 μg/ml polybrene (Merck Millipore TR-1003-G) three times and incubated at 32ºC. After 1 day, the infected cells were shifted to 37ºC and selected with puromycin. The knockdown efficiency was assessed by western blot or RT-PCR analysis.
The co-transfected cells were shifted to 32ºC. After 1 day, supernatants containing retroviruses were collected every 16-24 h three times. Cancer cell line (MKN45) was spin infected with the supernatants containing viruses for 90 min (1,455 × g) at 32ºC in the presence of polybrene (8 μg/ml) three times and incubated at 32ºC. After 1 day, the infected cells were shifted to 37ºC and selected with neomycin. The efficiency of stable expression was assessed by western blot or RT-PCR analysis.

RNA extraction, cDNA preparation, and qRT-PCR.
Total RNA was extracted from cancer cell lines or tumor tissues by the RNeasy Mini Kit (Qiagen 74106). cDNA synthesis was performed with 1 μg RNA using Prime Script First-Strand cDNA Synthesis Kit (Takara Bio RR036). The cDNA was quantified by real-time quantitative reverse transcription PCR (qRT-PCR) using SYBR Green (Applied Biosystems 4364344). The reaction was performed at 95 °C for 10 min, which was followed by a three-step PCR program of 95 °C for 30 s, 55 °C for 1 min, and 72°C for 30 s repeated for 50 cycles. The primers used in the qRT-PCR are listed in (Supplemental Table 1). The qRT-PCR was performed in triplicate for each sample. Quantitation was performed by the comparative Ct (2 -ΔΔCt ) method. The Ct value for each sample was normalized by the value for β-actin gene. Two or three independent experiments were analyzed statistically for differences in the mean values, and the P values are indicated in the figures.
Cell proliferation assay. Cells were plated at 1×10 4 cells per well in 6-well plates (SPL 30006) and cultured at 37°C incubator containing 5% CO2 for 7 days. At the indicated times, the number of cells in triplicate wells were determined using the trypan blue exclusion method (Thermo Fisher Scientific 15250-061).
Fatty acid oxidation assay. Fatty acid β-oxidation was measured with fatty acid oxidation complete assay kit (Abcam ab217602) according to the manufacturer's instruction. Briefly, cells were seeded into a black, clear-bottom cell culture plate (SPL 30296) at 5 × 10 4 cells per well and incubated overnight in 5% CO2 at 37 °C. The complete media were replaced with glucose deprivation media for 1 day or serum-free media for 2 days. After washing twice with pre-warmed fatty acid-free media, 90 μL fatty acid measurement media and 10 μL extracellular O2 consumption reagent (Abcam ab197243) were added to each well. Fatty acid measurement medium without cells was used as a signal control. Each well was sealed with 100 μL pre-warmed high sensitivity mineral oil. Signals were read immediately at 1.5 min intervals for 60 min using excitation/emission wavelengths of 380 nm/650 nm in EnVision Multilable Plate Reader (PerkinElmer). The plate was maintained at 37 °C throughout the course of the experiment. The data sets were analyzed statistically for differences in the mean values, and P values are indicated in the figures.
The extracellular acidification rate analysis. The extracellular acidification rate (ECAR) of cancer cell lines was determined using a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience). Cells were plated at 1×10 4 cells per well and cultured in complete media or serumfree media for 48 h. Prior to measurement, cells were incubated in unbuffered DMEM assay medium (Agilent 103334-100) in a non-CO2 incubator at 37 °C for 1 h. The DMEM assay medium for ECAR measurement contained 2 mM glutamine (Sigma-Aldrich G7513). The following compounds (Agilent 103020-100) were injected: glucose (10 mM), oligomycin A (1.5 µM), and 2-DG (100 mM). This allowed for calculation of glycolysis rate, glycolytic capacity, and glycolytic reserve. Basal ECAR was measured prior to addition of glucose.
Mitochondrial respiration analysis. The cellular oxygen consumption rate (OCR) of cancer cell lines was determined using a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience). Cells were plated at 1×10 4 cells per well and cultured in complete media for 48 h.
As a negative control, 50 μM reserpine (Sigma-Aldrich R0875) was added. After incubation, the cells were cooled on ice for 5 min and centrifuged at 100 x g for 8 min at 4°C. Hoechst-stained cells were resuspended in ice-cold HBSS media with 10 mM HEPES and 2% FBS, and added with 2 μg/ml of propidium iodide (PI) (Sigma-Aldrich P4170) to exclude dead cells. Side population cells were analyzed and sorted using a BD LSR-Fortessa X-20 Flow Cytometer (Becton Dickinson) and a BD FACSAria II Cell Sorter (Beckman Coulter), respectively. The Hoechst dye was excited at 350 nm and its fluorescence was measured at two wavelengths using a 450/20 nm band pass filter and a 675 nm long pass edge filter. A 610 nm short pass dichroic mirror was used to separate the emission wavelengths. Hoechst "Blue" represents the 450/20 nm filter, the standard analysis wavelength for Hoechst 33342 DNA content analysis. The far right of the Hoechst "Red" (the 675 nm filter) axis indicates cells positive for PI. A live gate of side population was determined on the flow cytometer using Hoechst blue and red axes to exclude dead cells, red cells (no Hoechst stain), and debris. Side population cells were also sorted and reanalyzed to establish high purity (> 98%).
Data were collected and analyzed using FlowJo software.
Spheroid formation assay. Cells were plated as single cell suspension at 1×10 4 cells per well in 6-well ultra-low attachment plates (Corning 3471) and cultured in serum-free DMEM/F12 medium (Gibco 11330-032) supplemented with B27 (Gibco 17504044), 20 ng/ml FGFb (invitrogen PHG0021), 20 ng/ml EGF (invitrogen PHG0311), and 1% penicillin/streptomycin. After two weeks, wells were analyzed for spheroid formation and were quantified using an inverted microscope. Spheroids were collected by centrifugation at 300 × g for 5 min and dissociated to singe cell suspension. The single cell suspension was plated at 1×10 4 cells per well in 6-well ultra-low attachment plates and cultured in serum-free conditioned media described above. After two weeks, wells were analyzed for subspheroid formation.
Spheroids with a diameter more than 50 μm were counted for the spheroid-forming index.    for the indicated times. Viperin protein was detected by immunoblot using MaP.VIP. αtubulin was used as a loading control. (B) MKN28 was treated with IFN-γ for 6 h and then added with S31-201, a STAT3 inhibitor at the indicated concentration for 24 h. Each protein was detected by immunoblot using specific monoclonal antibodies. Grp94 was used as a loading control. (C) MKN28 was cultured in hypoxia chamber for 24 h or treated with the hypoxia-mimetic agent CoCl2 for 8 h. The cells were treated with 2-ME at the indicated concentration. Each protein was detected by immunoblot using specific monoclonal antibodies. (D) MKN28 was cultured in the presence and absence of serum, and treated with LY 294002 or SF1670 at the indicated concentration for 24 h. Each protein was detected by immunoblot using specific monoclonal antibodies. Grp94 was used as a loading control. LY 294002, a PI3K/AKT inhibitor; SF1670, a PTEN inhibitor. (E) ChIP assay was performed for MKN28 cultured in serum-free media or hypoxia chamber for the indicated time. Chromatin samples were immunoprecipitated with a specific monoclonal antibody to HIF-1 and assessed by real-time PCR. Data are presented as means  SEM (n = 2 in triplicate). Statistical analysis was performed by t test (E). Note that HIF-1α did not bind to HRE2 of viperin promoter under serum starvation or hypoxia.  Spheroids of the stable cell lines were dissociated and counted. Single cell suspension was mixed with an equal volume of Matrigel. The mixture (1×10 3 cells/mouse) was injected subcutaneously into the flank of 6-week-old male nude mice (n = 6/cell line). Tumor growth was monitored weekly and tumor volume was measured using a metric caliper. (B) After 8 weeks, mice were sacrificed and tumors were isolated. Tumor size (top) and weight (bottom) were measured. Data are presented as means  SEM (n = 6). (C and D) Tumor growth in SP and non-SP cell-derived xenograft mouse models. (C) MKN28 cells expressing Luc control shRNA were stained with Hoechst 33342 and were sorted to SP and non-SP cells using flow cytometry. Single cell suspension of SP cells or non-SP cells and viperin KD whole cells was mixed with an equal volume of Matrigel. The mixture (1×10 4 cells/mouse) was injected subcutaneously into the flank of 6-week-old male nude mice (n = 6/cell line). Tumor growth was monitored weekly and tumor volume was measured using a metric caliper. (D) After 10 weeks, mice were sacrificed and tumors were isolated. Tumor size (top) and weight (bottom) were measured. Data are presented as means  SEM (n = 6). Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison test (A-D). *P < 0.05; **P < 0.01; ***P < 0.001. Figure 9. Working model of viperin-dependent cancer metabolic reprogramming and cancer progression. In normal condition, viperin is expressed in CSCs through the HIF-1 pathway and induces glycolysis and lipid metabolism to support maintenance of their properties (top). In the tumor microenvironment such as serum depletion, hypoxia, and IFN secretion, viperin is induced in both CSCs and non-CSCs through PI3K/AKT/mTOR/HIF-1 pathway and JAK/STAT pathway, and promotes glycolysis and lipid metabolism to enhance CSC properties, cancer cell proliferation, and cancer progression (bottom).