Fig 1.
KSHV-transformed cells have reduced levels of glucose consumption, lactate production, oxygen consumption and intracellular ATP.
(A). KMM cells have faster proliferation rate than MM cells. Cells were seeded at 4×104 cells/well in 24-well plates and counted daily without medium changes. (B). KMM but not MM cells form colonies in softagar. MM and KMM cells at 2x104 cells/well were plated in softagar in 6 well-plates for 14 days. Representative pictures captured at 40x magnification are presented in the left panel. (C-F). KMM cells have reduced levels of glucose consumption (C and D) and lactate production (E and F) measured by enzymatic assays in the medium (C and E) or adjusted for cell number (D and F). (G-H). KMM cells have reduced levels of intracellular ATP and oxygen consumption. Levels of intracellular ATP (G) and oxygen consumption (H) of MM and KMM were determined at day 2 post seeding. All data are presented as mean ± s.e.m. from three (n = 3, A, and C-G) or four (n = 4, H) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (B). * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 2.
KMM cells do not require glucose for proliferation, survival and formation of colonies in Softagar.
(A). KMM cells do not require glucose for proliferation. MM and KMM cells seeded at 105 cells/well in 6-well plates in complete media (25 mM glucose) which were replaced with glucose-free or complete medium the following day, and cell numbers were counted daily. (B). KMM cells do not require glucose for formation of colonies in softagar. MM and KMM cells were plated in softagar in the presence or absence of glucose as described in Fig 1B. Representative pictures captured at 40x magnification are presented in the left panel. Colonies with diameter >50 μm were counted and colony numbers in each field are presented in the right panel. (C-D). MM but not KMM cells are cell cycle arrested following glucose deprivation. Cell cycle distribution and BrdU incorporation were analyzed by flow cytometry following 24 h glucose deprivation. (E). Glucose deprivation induces apoptosis in MM but not KMM cells. Apoptotic cells were detected by Annexin V staining following 48 h of glucose deprivation. (F). Intracellular ATP levels decrease in MM but not KMM cells following glucose deprivation. Intracellular ATP levels were determined in cells grown in media with or without glucose at day 2 and 3. All data are presented as mean ± s.e.m. from three (n = 3) independent experiments, each with three repeats. NS, not significant; *** P < 0.001.
Fig 3.
KSHV miRNAs and vFLIP mediate inhibition of glucose consumption, aerobic glycolysis and oxidative phosphorylation, as well as glucose-independent cell proliferation and cellular transformation.
(A). Cell proliferation of MM cells and those infected by recombinant KSHV including wild-type (KMM), and mutants with a deletion of a cluster of 10 precursor miRNAs (ΔmiRs), vFLIP (ΔvFLIP) or vCyclin (ΔvCyclin) under normal culture conditions as described in Fig 1A. (B-E). Levels of glucose consumption (B), lactate production (C), intracellular ATP (D) and oxygen consumption (E) of cells infected by different recombinant viruses determined as described in Fig 1C–1H. (F). Cell proliferation of cells infected by different recombinant viruses in the presence or absence of glucose measured as described in Fig 2A. (G-I). Cell cycle profiles (G), BrdU incorporation (H) and apoptosis (I) of cells infected by different recombinant viruses measured as described in Fig 2C–2E. (J). Formation of colonies in softagar of cells infected by different recombinant viruses in the presence or absence of glucose measured as described in Fig 2B. All data are presented as mean ± s.e.m. from three (n = 3, A-D, and F-J) or four (n = 4, E) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (J). NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 4.
KSHV vFLIP and the miRNA cluster mediate KSHV downregulation of GLUT1 and GLUT3.
(A-C). Analysis of GLUT1 and GLUT3 expression in MM and KMM cells by RT-qPCR (A), Western-blot (B) and flow cytometry (C). (D-F). Analysis of GLUT1 and GLUT3 expression in cells infected by different recombinant viruses by RT-qPCR (D), Western-blot (E) and flow cytometry (F). β-actin and β-tubulin were used as internal controls for RT-qPCR and Western-blot, respectively. For panels C and F, the Y-axis is shown as normalized cell numbers. All data are presented as mean ± s.e.m. from three (n = 3, A and D) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (B, C, E, and F). NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 5.
Knock down of RelA increases the expression of GLUT1 and GLUT3, and aerobic glycolysis.
(A). Analysis of RelA, GLUT1 and GLUT3 proteins in MM and KMM cells by Western-blot following knock down of RelA. Detection of RelA, GLUT1 and GLUT3 proteins in MM and KMM cells following transfection with a siRNA to RelA (siRelA) or a scrambled control (Scr) for 3 days. β-tubulin was used as an internal control for loading. (B-C). Analysis of RelA (B), GLUT1 (C) and GLUT3 (D) mRNAs in MM and KMM cells by RT-qPCR following knock down of RelA. Cells were treated as described in (A). β-actin was used as an internal control for qPCR. (E). Knock down of RelA decrease cell proliferation of KMM but not MM cells. Cell proliferation were examined following knock down of RelA. (F-G) Knock down of RelA increases glucose consumption (F) and lactate production (G) in KMM cells. Glucose consumption and lactate production were determined as described in Fig 1C and 1E following knock down of RelA. Experiments were repeated three times, each with three repeats and representative results were presented. * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 6.
The NF-κB pathway mediates KSHV suppression of GLUT1 and GLUT3, and aerobic glycolysis.
(A-B). Inhibition of the NF-κB pathway increases the expression of GLUT1 and GLUT3. MM and KMM cells were treated with NF-κB inhibitors JSH23 (30 μM) or BAY 11–7082 (2 μM), and examined for the expression of GLUT1 and GLUT3 expression by RT-qPCR at 8 h post-treatment (A) and flow cytometry at 24 h post-treatment (B). Y-axis in (B) is shown as normalized cell numbers. β-actin was used as internal controls for qPCR. (C-D). Inhibition of the NF-κB pathway increases glucose consumption (C) and lactate production (D). MM and KMM cells were treated with NF-κB inhibitor JSH23 (30 μM) and glucose consumption and lactate production were determined as described in Fig 1C and 1E. (E-F). Inhibition of the NF-κB pathway reduces cell proliferation and inhibits cellular transformation. Cell proliferation (E) and colony formation in softagar (F) were examined in the presence of JSH23 (30 μM) as described in Fig 1A and 1B. (G-H). Inhibition of the NF-κB pathway increases apoptosis and reduces BrdU incorporation. Apoptosis (G) and BrdU incorporation (H) of MM and KMM cells were examined following treatment of JSH23 (30 μM) for 48 h as described in Fig 2D and 2E. All data are presented as mean ± s.e.m. from three (n = 3, A, and C-H) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (B and F). * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 7.
Overexpression of GLUT1 or GLUT3 enhances aerobic glycolysis, and sensitizes KMM cells to apoptosis upon glucose deprivation.
(A-B). Stable expression of GLUT1 and GLUT3 in MM and KMM cells. Cells with stable expression of GLUT1 or GLUT3 were analyzed by Western-blot (A) and flow cytometry (B). Y-axis in (B) is shown as normalized cell numbers. β-tubulin was used as internal controls for Western-blot. (C-D). Overexpression of GLUT1 or GLUT3 increases glucose consumption (C) and lactate production (D). MM and KMM cells with stable expression of GLUT1 or GLUT3 were examined for glucose consumption and lactate production as described in Fig 1C and 1E. (E-G). Overexpression of GLUT1 or GLUT3 sensitizes KMM cells to glucose deprivation shown in cell proliferation (E) and apoptosis (F) but not cell cycle distribution (G). Cell proliferation, apoptosis and cell cycle progression of MM and KMM cells stably expressing GLUT1 or GLUT3 were examined in the presence or absence of glucose as described in Fig 2A, 2C and 2E. (H). Overexpression of GLUT1 or GLUT3 reduces the efficiency of colony formation of KMM cells in softagar. Colony formation of KMM cells was examined in the presence or absence of glucose as described in Fig 1A. All data are presented as mean ± s.e.m. from three (n = 3, C-H) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (A, B, and H). NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 8.
Overexpression of GLUT1 or GLUT3 impairs the AKT-NF-κB pro-survival pathway.
(A). Effect of GLUT1 and GLUT3 on the AKT-NF-κB pro-survival pathway. Cell lysates from KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells cultured under normal conditions were analyzed by Western-blot with the specified antibodies. β-tubulin was used as internal controls. (B-D). Overexpression of GLUT1 or GLUT3 increases autophagy in KMM cells. KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells transduced with mCherry-LC3 for 48 h were examined for the formation of LC3 punctate. Representative images were captured at 1,000x magnification using laser-scanning confocal microscopy (B). The percentage of cells with LC3 punctate (C) and the number of LC3-positive dots per cell (D) were counted. (E). Glucose deprivation and inhibition of PI3K reduce NF-κB activation. Total and phosphorylation of NF-κB in KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells in the presence or absence of glucose as well as with and without treatment with 12.5 μM PI3K inhibitor LY294002 were examined by Western-blot at 48 h post-treatment. (F). Glucose deprivation and inhibition of PI3K sensitize KMM cells to apoptosis. KMM-vector, KMM-GLUT1 and KMM-GLUT3 cells in the presence or absence of glucose as well as with and without treatment with 12.5 μM PI3K inhibitor LY294002 were examined for apoptosis at 72 h post-treatment. All data are presented as mean ± s.e.m. from three (n = 3, C, D, and F) independent experiments, each with three repeats. Representative images from three independent experiments with similar results are presented (A, B, and E). NS, not significant; *** P < 0.001.
Fig 9.
GLUT1 and GLUT3 are downregulated in KSHV-infected cells in human KS tumors.
(A-B). Representative illustration of dual immunofluorescence detection of LANA and GLUT1 (A) or GLUT3 (B) in a normal human skin section and a KS tumor section. The tissue sections were counterstained with DAPI. Magnifications 600x. Boxed areas are enlarged. (C) Analysis of GLUT1 expression in LANA negative (-) and LANA positive (+) cells in KS tumors (n = 25). (D) Analysis of GLUT3 expression in LANA negative (-) and LANA positive (+) cells in KS tissues (n = 17). (E-F) Negative correlation of the average percentage of LANA-positive cells with the average expression level of GLUT1 (E) and GLUT3 (F) in KS tumors. For (C and D), the boxes represent the interquartile range (25-75th centiles. The horizontal line inside the box indicates the median. The vertical whiskers extend to the maximum and minimum values. Statistical analysis was performed by Wilcoxon matched-pairs signed-ranks test. Expression levels of GLUT1 and GLUT3 were quantified based on immunofluorescence staining, using a modified His-score as described in the Materials and Methods. **P < 0.01; ***P < 0.001.
Fig 10.
Suppression of aerobic glycolysis in PEL cells.
(A). Downregulation of GLUT1 in PEL cell lines. Western-blot detection of GLUT1 and GLUT3 protein levels in PEL cell lines BCBL1, BC3 and BCP1, and uninfected and KSHV-infected BJAB cells. (B). Cell proliferation rates of PEL cell lines BCBL1, BC3 and BCP1, and uninfected and KSHV-infected BJAB cells. (C-D). KSHV-infected cells have reduced levels of glucose consumption (C) and lactate production (D). Glucose consumption and lactate production were determined as described in Fig 1C and 1E. Experiments were repeated three times, each with three repeats and representative results were presented. *** P < 0.001.
Fig 11.
A model illustrates that vFLIP and the miRNA cluster mediate KSHV suppression of aerobic glycolysis to promote cell survival by downregulating the expression of GLUT1 and GLUT3 in an AKT-NF-κB-dependent manner.