Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells

Abstract

Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.

Fig. 1.

KSHV infection induces aerobic glycolysis. (A) Glucose uptake is increased in KSHV- versus mock-infected cells. Forty-eight hours after infection, mock (M) or KSHV (K) infected TIME cells were exposed to a radiolabeled glucose analog (Amersham TRL383) for 10 min followed by intracellular quantification of radioactivity. Cells were treated with Dfo (D), where indicated for the last 16 h of infection. KSHV-infected cells were greater than 90% latently infected and less than 1% of the cells expressed lytic markers. (B) Western blot analysis of GLUT3 expression in mock- and KSHV-infected TIME cells. Cells were treated with Dfo (D) where indicated for the last 8 h of infection. (C) Western blot analysis of HK2 expression in mock- and KSHV-infected TIME cells 48 h postinfection. (D) Increased lactate production in KSHV- versus mock-infected TIME cells. Cellular supernatant was collected from mock- or KSHV-infected cells 48 h postinfection and lactate production was quantified by an enzymatic colorimetric assay.

Fig. 2.

KSHV-infected cells increase lactic acid production and decrease oxygen consumption. Seahorse Bioscience extracellular flux analyzer was used to measure the ECAR (A and B) and OCR (C and D) in mock- and KSHV-infected TIME (A and C) or 1° hDMVECs (B and D). Forty-eight hours postinfection, cells were seeded in 24-well plates in unbuffered solution, the wells were sealed by plungers, and ECAR and OCR was measured over 4 min. The wells were then released and resealed and measured again (each datapoint is a separate measurement of rate for quadruplicate samples). A and C are from the same experiment and B and D are from the same experiment. Line A indicates the injection time of oxamate (100 mM) and line B indicates the injection time of rotenone (1 μM).

Fig. 3.

Overview of cellular metabolism with glycolytic and mitochondrial inhibitors. 2DG inhibits glycolysis by metabolic trapping. Oxamate inhibits LDH activity. Rotenone inhibits complex I of mitochondrial oxidative phosphorylation.

Fig. 4.

Inhibitors of glycolysis selectively induce apoptosis in KSHV latently infected cells. (A) Mock- (blue) and KSHV- (pink) infected TIME cell death percentages were determined by a Trypan blue exclusion assay. At 48 h postinfection, cells were seeded into flasks at equal numbers and treated for an additional 48 h with 0, 160, and 320 mM 2DG. Cells death rates were determined by counting cells using a hemocytometer after addition of Trypan blue. Cell death rate (%) = no. of dead cells/no. of total cells. (B) At 48 h postinfection, mock- and KSHV-infected TIME (Upper) cells or 1° hDMVECs (Lower) were treated with 0 and 100 mM oxamate for an additional 48 h and cell death was measured as in A. (C) Forty-eight hours postinfection, mock- and KSHV-infected TIME cells were treated with 0 or 100 mM oxamate for 36 h and then treated with Image-It Dead Green Viability stain for 15 min. (D) Western blot analysis of caspase-3 and PARP cleavage after treatment of mock- (M) and KSHV- (K) infected cells with oxamate for 36 h. St = 1 μM stauroporin treatment of mock-infected cells (8 h). (E) Mock- and KSHV-infected TIME-cell death percentages were determined by Trypan blue assay (as in A and B). Cells were treated for 36 h with 0 mM oxamate (control), 100 mM oxamate, or 100 mM oxamate + 100 uM ZVAD (caspase inhibitor).