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. 2020 Apr 7;117(14):8083-8093.
doi: 10.1073/pnas.1918607117. Epub 2020 Mar 25.

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis

Un Yung Choi  1 , Jae Jin Lee  1 , Angela Park  1 , Wei Zhu  2 , Hye-Ra Lee  3 , Youn Jung Choi  1 , Ji-Seung Yoo  4 , Claire Yu  2 , Pinghui Feng  1   5 , Shou-Jiang Gao  1   6   7 , Shaochen Chen  2 , Hyungjin Eoh  8 , Jae U Jung  8
Affiliations

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis

Un Yung Choi et al. Proc Natl Acad Sci U S A. .

Abstract

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.

Keywords: K1; Kaposi's sarcoma-associated herpesvirus (KSHV); cancer metabolism; proline metabolism; pyrroline-5-carboxylate reductase (PYCR).

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Conflict of interest statement

Competing interest statement: J.U.J. is a scientific advisor of Vaccine Stabilization Institute, a California corporation.

Figures

Fig. 1.
Fig. 1.
KSHV induces nonessential amino acid metabolism in 3D culture conditions. (A) Proliferation rate of uninfected or KSHV-infected TIME cells in 2D monolayer. Proliferation was measured by counting the cell numbers. Data are presented as the mean ± SEM. (B) Representative pictures (Left) and size quantification (Right) of 3D spheroids of uninfected or KSHV-infected TIME cells cultured in ultralow attachment condition with 4% Matrigel for 8 d. (Scale bars: 100 μm.) Data are presented as the mean ± SEM *P < 0.05, by Student’s t test. (C) Proliferation rate of uninfected or KSHV-infected MCF10A cells in 2D monolayer. Proliferation was measured by counting the cell numbers. Data are presented as the mean ± SEM. (D) Representative images of uninfected or KSHV-infected MCF10A cells cultured on Matrigel for 14 d. Acini were stained with the nuclei counterstained with Hoechst 33342 (blue) and Ki67 antibody (red). GFP signal was from the BAC16 KSHV genome. (Scale bars: 100 μm, Left.) Quantitation of Ki67 positive cells in acini as box-and-whisker plot. The number of biological replicates for each experiment was n ≥ 3 (Right). *P < 0.05, by Student’s t test. (E) Scatterplots of KSHV-induced metabolic pathways in TIME cells and MCF10A cells. KSHV-induced metabolic pathways in TIME and MCF10A cell types are annotated in the graph. Node color is based on its P value, and the node size is based on pathway impact score.
Fig. 2.
Fig. 2.
K1 interacts with and enhances PYCR enzyme activity. (A) Purification of K1(C) binding proteins. Arrowheads denote the bands that were excised and subjected to MS-based analysis for protein identification. (B) A series of K1 expression constructs (Left) were transfected into HEK293 cells and cell extracts were immunoprecipitated with an anti-K1 antibody, followed by immunoblotting with the indicated antibodies (Right). (C) iSLK-BAC16 KSHV WT and iSLK-BAC16 ΔK1 (ΔK1) cells were stimulated with doxycyline (Dox, 1 μg/mL) and sodium butyrate (NaB, 1 mM) to induce viral reactivation. Cell extracts were immunoprecipitated with an anti-K1 antibody, followed by immunoblotting with the indicated antibodies. (D) Representative confocal fluorescence images of K1. HeLa cells expressing K1 were stained with anti-K1 and anti-mitofilin antibodies. Merged images show K1 (green), mitofilin (red), and nucleus (blue). (Scale bars: 10 μm.) (E) Purification of recombinant PYCR2 complex from E. coli. The arrowhead indicates the His-PYCR2 and the asterisks indicate GST and GST-K1. As a control, the purified recombinant GST protein was added to His-PYCR2. (F) PYCR2 enzymatic reaction. (G) Enzyme activity plots of purified His-PYCR2 with GST or GST-K1. Right graph shows Michaelis–Menten model for His-PYCR2 enzyme activity with GST or GST-K1. Data are presented as the mean ± SD. (H) Enzyme activity plots of purified His-PYCR2 with GST or GST-K1 in presence of proline and ATP. Data are presented as the mean ± SD. (I) MS-based metabolomics analysis of intracellular amino acids. HEK293T cells were collected 48 h after transfection with mock or K1 expression vector, and the amounts of intracellular amino acid were determined by LC-MS (n = 5). Log2 fold change values (K1/mock) data are presented as the mean ± SEM. **P < 0.01 and ***P < 0.001, by Student’s t test.
Fig. 3.
Fig. 3.
K1 promotes cell growth in 3D spheroid culture system. (A) Representative pictures and size quantification of 3D spheroids of mock-, K1 WT-, or mutant-expressing TIME and HFF cells cocultured in ultralow attachment condition. (Scale bars: 200 μm.) *P < 0.05, by one-way ANOVA. (B) A 3D confocal microscopy image of R-TIME-cultured hydrogel scaffold. Bottom shows the average of total fluorescence intensity at day 9 normalized to day 2 (seeding amount). *P < 0.05 and **P < 0.01, by one-way ANOVA. The 3D reconstruction and analysis of fluorescence intensity from multiple z-series images were performed using ImageJ. (C) Representative pictures and size quantification of 3D spheroids of mock-, K1 WT-, or mutant-expressing MCF10A cells. (D) MDA-MB-231 cells cultured in ultralow attachment condition. (Scale bars: 200 μm.) *P < 0.05; **P < 0.01; ****P = 0.0001, by one-way ANOVA. (E) ATP production of MCF10A cells expressing mock, K1 WT, or mutant. *P < 0.05, by one-way ANOVA. (F) Representative pictures (Upper) and size quantification (Lower) of 3D spheroids of uninfected, KSHV WT-infected, or KSHV K1 ΔC-infected or KSHV K1 TYF-infected TIME cells cultured in Matrigel for 12 d. (Scale bars: 100 μm.) The number of biological replicates for each experiment was n ≥ 3. **P = 0.0001, by one-way ANOVA. (G) Representative images of uninfected, KSHV WT-infected, or KSHV K1 ΔC-infected or KSHV K1 TYF-infected MCF10A cells cultured on Matrigel for 21 d. (Upper) Acini were stained with the nuclei counterstained with Hoechst 33342 (blue) and Ki67 antibody (red). GFP is from the BAC16 KSHV genome. (Scale bars: 100 μm.) (Lower) Quantification of Ki67 positive cells in acini. The number of biological replicates for each experiment was n ≥ 3. **P < 0.01 and ****P = 0.0001; ns, not significant, by one-way ANOVA.
Fig. 4.
Fig. 4.
K1 induces collagen synthesis and reduces ROS in 3D spheroids. (A) Representative confocal image of collagen I (red) and K1 (green) immunofluorescence on mock-, K1 WT-, or mutant-expressing TIME and HFF cell cocultured spheroids in ultralow attachment condition. Nuclei were stained with Hoechst 33342 (blue). (Scale bars: 10 μm.) (B) Quantification of collagen I signal intensity in 3D spheroids. Collagen intensity of spheroids was divided by areas of spheroids. Data are mean ± SEM. **P < 0.01 and ****P = 0.0001, by one-way ANOVA. (C) Intracellular ROS level in mock-, K1 WT-, or mutant-expressing TREX 293T cell spheroids. a.u., arbitrary units. Data are mean ± SEM. *P < 0.05; ns, not significant, by one-way ANOVA.
Fig. 5.
Fig. 5.
PCYR expression and interaction are essential for K1-mediated transformation. (A) Representative pictures of mock-, K1 WT-, or mutant-expressing TIME cells treated with scramble shRNA (scramble) or PYCR-specific shRNA (shPYCR1/2) in ultralow attachment condition. Right graph shows size quantification of the spheroids. (Scale bars: 200 μm.) *P < 0.05, by two-way ANOVA. (B) Scramble- or shPYCR-treated KSHV-infected TIME cells were subjected to low-density culture (1,500 cells per six-well plates) for clonogenic assay. Representative pictures for colony growth are shown (Upper). Quantification of the number of colonies is shown (Lower). Data are presented as the mean ± SD. ****P < 0.0001, by Student’s t test. (C) Representative pictures and size quantification of 3D spheroids of scramble- or shPYCR-treated KSHV-infected TIME cells cultured in ultralow attachment condition with 4% Matrigel. (Scale bars: 100 μm.) Data are presented as the mean ± SEM. ****P < 0.0001, by Student’s t test. (D) Scramble- or shPYCR-treated KMM cells were subjected to low-density culture (1,000 cells per six-well plates) for clonogenic assay. Representative pictures for colony growth are shown (Upper). Quantification of the number of colonies is shown (Lower). Data are presented as the mean ± SD. ****P = 0.0001, by one-way ANOVA. (E) Representative pictures and size quantification of 3D spheroids of scramble control- or shPYCR-treated KMM cells cultured in ultralow attachment condition. (Scale bars: 400 μm.) Data are presented as the mean ± SEM. ****P = 0.0001, by one-way ANOVA. (F) Intracellular ATP levels of scramble control or shPYCR KMM cells are seeded in ultralow attachment condition. Intracellular ATP levels were measured as an indicator of cell viability using CellTiter-Glo reagents after 4 d of shRNA treatment. Data are presented as the mean ± SD. The number of biological replicates for each experiment was n ≥ 3. ****P = 0.0001, by one-way ANOVA.
Fig. 6.
Fig. 6.
Proline metabolic differences between in vivo tumors. (A) MDA-MB-231 cells (1 × 106 cells) expressing mock, K1 WT, or mutants were injected s.c. into nude mice (n = 9 to 10). Tumor volume was plotted as indicated. Data are presented as the mean ± SEM. **P < 0.01 and ****P = 0.0001, by two-way ANOVA. At 11 d after injection, xenograft tumors were (B) harvested and (C) weighed. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by one-way ANOVA. (D) Metabolite abundance of proline metabolism pathway in mock-, K1 WT-, or mutant-expressing MDA-MB-231 tumors measured by LC-MS. Levels of metabolites are related to proline synthesis. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01, by one-way ANOVA.
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References

    1. Langhans S. A., Three-Dimensional in vitro cell culture models in drug discovery and drug repositioning. Front. Pharmacol. 9, 6 (2018). - PMC - PubMed
    1. Pampaloni F., Reynaud E. G., Stelzer E. H., The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839–845 (2007). - PubMed
    1. Elia I., et al. , Proline metabolism supports metastasis formation and could be inhibited to selectively target metastasizing cancer cells. Nat. Commun. 8, 15267 (2017). - PMC - PubMed
    1. Mantilla B. S., et al. , Proline metabolism is essential for trypanosoma brucei brucei survival in the tsetse vector. PLoS Pathog. 13, e1006158 (2017). - PMC - PubMed
    1. Toyoshima K., et al. , Increased plasma proline concentrations are associated with sarcopenia in the elderly. PLoS One 12, e0185206 (2017). - PMC - PubMed

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