Oncogenic Kaposi's Sarcoma-Associated Herpesvirus Upregulates Argininosuccinate Synthase 1, a Rate-Limiting Enzyme of the Citrulline-Nitric Oxide Cycle, To Activate the STAT3 Pathway and Promote Growth Transformation

Abstract

Cancer cells are required to rewire existing metabolic pathways to support their abnormal proliferation. We have previously shown that, unlike glucose-addicted cancers, Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells depend on glutamine rather than glucose for energy production and amino acid and nucleotide syntheses. High-level consumption of glutamine is tightly regulated and often coupled with the citrulline-nitric oxide (NO) cycle. We have found that KSHV infection accelerates nitrogen efflux by upregulating the expression of argininosuccinate synthase 1 (ASS1), a key enzyme in the citrulline-NO cycle. KSHV utilizes multiple microRNAs to upregulate ASS1 expression. Depletion of either ASS1 or inducible nitric oxide synthase (iNOS) in KSHV-transformed cells suppresses growth proliferation, abolishes colony formation in soft agar, and decreases NO generation. Furthermore, by maintaining intracellular NO levels, ASS1 expression facilitates KSHV-mediated activation of the STAT3 pathway, which is critical for virus-induced transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote growth transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.IMPORTANCE We have previously shown that Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells depend on glutamine rather than glucose for energy production and amino acid and nucleotide syntheses. In this study, we have further examined how the KSHV-reprogramed metabolic pathways are regulated and discovered that KSHV hijacks the citrulline-nitric oxide (NO) cycle to promote growth proliferation and transformation. Multiple KSHV-encoded microRNAs upregulate argininosuccinate synthase 1 (ASS1), a key enzyme in the citrulline-NO cycle. ASS1 is required for KSHV-induced proliferation, colony formation in soft agar, and NO generation of KSHV-transformed cells, which also depends on inducible nitric oxide synthase. By maintaining intracellular NO levels, ASS1 mediates KSHV activation of the STAT3 pathway, which is essential for KSHV-induced abnormal cell proliferation and transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote growth transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.

Keywords: ASS1; KSHV; Kaposi’s sarcoma; NO; STAT3; argininosuccinate synthase 1; citrulline-nitric oxide cycle; iNOS; inducible nitric oxide synthase; microRNA; nitric oxide.

FIG 1

Multiple KSHV-encoded miRNAs mediate upregulation of ASS1 expression. (A) The ASS1 transcript is upregulated in KSHV-transformed cells. RT-qPCR was used to examine the level of the ASS1 transcript. (B) Validation of the specificity of ASS1 antibody. Western blotting was used to examine MM and KMM cells stably expressing ASS1 or a vector control. The intensity of the upper band was dramatically increased in cells expressing ASS1 but not the vector control, indicating that the upper band is specific for the ASS1 protein. (C) ASS1 protein is upregulated in KSHV-transformed cells. Western blotting was used to examine the level of ASS1 protein. (D and E) ASS1 transcript and protein are upregulated in KSHV-infected cells. RT-qPCR and Western blot results show that the expression of ASS1 was upregulated at both the mRNA and protein levels in iSLK-KSHV cells (D) and BJAB-KSHV cells (E) compared to uninfected cells and in PEL cells compared to BJAB cells (E). (F and G) The KSHV miRNA cluster is required for the upregulation of ASS1 expression. Shown are data from analysis of ASS1 expression in cells infected by different recombinant viruses, including wild-type KSHV (KMM) and a mutant with a deletion of a cluster of 10 pre-miRNAs (ΔmiRs), vFLIP (ΔvFLIP), or vCyclin (ΔvCyclin), by RT-qPCR (F) and Western blotting (G). (H and I) Expression of the KSHV miRNA cluster is sufficient to upregulate ASS1 expression. ASS1 expression was detected by Western blotting (H) and RT-qPCR (I) in MM cells and ΔmiRs cells overexpressing the KSHV miRNA cluster or the control empty vector pITA (MM+pITA). (J) Expression of multiple individual miRNAs is sufficient to upregulate ASS1 expression. ASS1 protein was detected by Western blotting in ΔmiRs cells overexpressing individual miRNAs. β-Actin and β-tubulin were used as internal controls for RT-qPCR and Western blotting, respectively. ** and *** represent P values of <0.01 and <0.001, respectively, while NS indicates not significant.

FIG 2

ASS1 is essential for KSHV-induced cell proliferation and cellular transformation. (A and B) Analysis of ASS1 expression in MM and KMM cells following depletion of ASS1 expression by RT-qPCR (A) and Western blotting (B). (C and D) ASS1 knockdown inhibits cell proliferation and cellular transformation of KMM cells. Cell proliferation (C) and colony formation in soft agar (D) were examined following knockdown of ASS1 with 3 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (control [ctl]). Representative pictures at a ×40 magnification from soft agar assays are shown (D, left). Colonies with diameters of >50 μm were counted, and the relative number of colonies per field was quantified (D, right). (E and F) ASS1 knockdown induces cell cycle arrest and apoptosis. (E) The cell cycle was analyzed by flow cytometry 48 h following transduction with ASS1 shRNAs (shASS1). (F) Apoptotic cells were detected by annexin V staining 72 h following transduction with ASS1 shRNAs. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively, while NS indicates “not significant.”

FIG 3

iNOS is essential for KSHV-induced cell proliferation and cellular transformation. (A) Examination of eNOS, iNOS, or nNOS expression by RT-qPCR in MM and KMM cells. (B and C) Analysis of iNOS expression in MM and KMM cells following iNOS knockdown by RT-qPCR (B) and Western blotting (C). (D and E) iNOS knockdown inhibits cell proliferation and cellular transformation of KMM cells. Cell proliferation (D) and colony formation in soft agar (E) were determined following depletion of iNOS expression with 3 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl). Representative pictures at a ×40 magnification from soft agar assays are shown (E, left). Colonies with diameters of >50 μm were counted, and the results were graphed (E, right). (F and G) iNOS knockdown induces cell cycle arrest and apoptosis. (F) The cell cycle was analyzed by flow cytometry 48 h following transduction with iNOS shRNAs. (G) Apoptotic cells were detected by annexin V staining 72 h following transduction with ASS1 shRNAs. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively, while NS indicates “not significant.”

FIG 4

Detection of intracellular NO levels with DAR is not altered by different intracellular ROS levels. (A) Chemical equation showing that the reaction of DAR with NO requires ROS. (B) MM cells have higher intracellular ROS levels than KMM cells. (C) Flow cytometry analysis showing that the NO donor SNAP does not alter intracellular ROS levels in MM and KMM cells. APC-A indicates allophycocyanin (APC) fluorescent intensity.

FIG 5

Silencing of ASS1 or iNOS expression reduces the intracellular production of NO. (A and B) Detection of NO with DAR in MM and KMM cells following knockdown of ASS1 with 3 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl). The intracellular NO level was examined with a fluorescence microscope (A) and quantified for relative intensity with ImageJ (B). (C and D) Detection of NO with DAR in MM and KMM cells following knockdown of iNOS with 3 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl). The intracellular NO level was detected with a fluorescence microscope (C), and the relative intensity was quantified with ImageJ (D). CTCF represents the corrected total cell fluorescence by ImageJ. * and ** represent P values of <0.05 and <0.01, respectively, while NS indicates “not significant.”

FIG 6

The iNOS inhibitor L-NAME reduces intracellular NO levels and inhibits cell proliferation of MM and KMM cells. (A) Detection of intracellular NO levels with DAR in MM and KMM cells with or without treatment with the NO donor L-NAME. Representative images were captured at a ×20 magnification with a fluorescence microscope. (B) Quantification of intracellular NO levels in MM and KMM cells with ImageJ. CTCF represents the corrected total cell fluorescence by ImageJ. (C) Inhibition of cell proliferation of MM and KMM cells by L-NAME. Numbers of MM and KMM cells treated with 0, 4, 6, or 8 mM L-NAME were counted over time. * and *** represent P values of <0.05 and <0.001, respectively, while NS indicates “not significant.”

FIG 7

NO mediates ASS1 and iNOS induction of STAT3 activation. (A) Depletion of ASS1 expression inhibits STAT3 tyrosine phosphorylation. MM and KMM cells were transduced with 3 ASS1 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl) and examined for the levels of total and phosphorylated STAT3 (Y705) by Western blotting. The ASS1 protein level was also examined to monitor knockdown efficiencies, while β-tubulin was used as a loading control. (B and C) The NO donor SNAP increases intracellular NO levels. MM and KMM cells were treated with 0.5 mM SNAP for 1 h and examined for intracellular NO by DAR staining. The intracellular NO level was examined with a fluorescence microscope (B), and the relative intensity was quantified with ImageJ (C). (D) ASS1 knockdown has no effect on ROS production. MM and KMM cells transduced with 3 ASS1 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl) for 2 days were examined for intracellular ROS levels. (E) The NO donor SNAP partially rescues STAT3 activation following ASS1 knockdown. MM and KMM cells transduced with an ASS1 shRNA (shRNA3) or a scrambled shRNA (ctl) for 2 days were treated with 0.5 mM SNAP for 0.5 h and then examined for the levels of total and phosphorylated STAT3 (Y705) by Western blotting. β-Tubulin was used as a loading control. APC-A indicates allophycocyanin (APC) fluorescent intensity.

FIG 8

ASS1 does not regulate the expression of GLUT1 and GLUT3, and glucose deprivation does not affect ASS1 protein and intracellular NO levels. (A) ASS1 knockdown has no effect on the expression of GLUT1 and GLUT3 proteins. MM and KMM cells transduced with 3 ASS1 shRNAs (shRNA1, -2, or -3) or a scrambled shRNA (ctl) for 2 days were examined for the levels of GLUT1 and GLUT3 proteins. (B) Overexpression of ASS1 has no effect on the expression of GLUT1 and GLUT3 proteins. MM and KMM cells overexpressing ASS1 or a vector control (V) for 2 days were examined for the levels of GLUT1 and GLUT3 proteins. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C and D) Glucose deprivation does not affect ASS1 protein and intracellular NO levels. MM and KMM cells were seeded overnight in full medium, cultured in medium with and without glucose for 12 and 24 h, and examined for the level of ASS1 protein (C) or intracellular NO (D).

FIG 9

Working model illustrating that KSHV hijacks the citrulline-NO cycle to promote KSHV-induced cell proliferation and cellular transformation. Multiple KSHV miRNAs upregulate ASS1 expression to maintain intracellular NO levels and STAT3 activation, which is essential for KSHV-induced cell proliferation and cellular transformation.