Elevated iNOS and 3'-nitrotyrosine in Kaposi's Sarcoma tumors and mouse model

Abstract

Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.

Keywords: HHV8; Kaposi's Sarcoma; Kaposi's Sarcoma associated herpesvirus; L-NMMA; LANA; Nitrotyrosine; iNOS.

Fig. 1

iNOS is expressed at high-levels in KS. A. Analysis of KS transcriptomics (NCBI GEO GSE147704) reveals NOS2/iNOS is among the most up-regulated gene transcripts in KS lesions. Strongly down-regulated genes are also indicated. B–C. RT-qPCR for control skin (Ctrl, grey) or KS lesion (Lesion, red) for patient samples 3128, 3129, C034. Primers specific for iNOS, ORF75, and were analyzed by ΔΔCT method relative to GAPDH. Error bars are standard deviation from mean. *p < .05, **p < .01, ****p < .001 using 2way ANOVA with Fisher's Least Significant Difference test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

iNOS is enriched in LANA⁺ KS tumor cells. A. IHC of normal skin (left) or KS tumor (right) tissue sections stained with anti-LANA (top) or anti-iNOS (bottom). Enlarged image of KS samples (right panels). B. IF of normal skin (top) or KS tumor (bottom) with antibodies for iNOS (green), LANA (red), or DAPI (blue)/merge. Scale bar = 10 μm. C. Quantification of the percentage of LANA⁺ cells in sections examined from patient biopsy samples. D. IF of LANA negative and LANA positive nuclei from KS lesions showing iNOS (green) and LANA (red), and DAPI/merge. E. Quantification of IF images represented in panel D, for four different patient biopsies. Each dot represents the mean value for each sample. Mean values were measured by manual counting from five different regions of the histological section. Percent cells with iNOS signals were scored for their colocalization with LANA⁺ or LANA^- cells. *p < .05, **p < .01, ****p < .0051 one-way ANOVA (C), and Mann-Whitney, unpaired U test (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Detection of 3-nitrotyrosin in KS tissue sections. A. Schematic of 3-nitrotyrosine formation by iNOS and NO metabolism. B. IF images of 3-nitrotyrosine (green) or LANA (red) and merged with DAPI (blue) in normal skin (top panel) or KS lesions KS-1 (KSC3094) or KS-2 (KS2005961) (bottom panels). Yellow arrow indicates colocalization of 3-nitrotyrosine with LANA-NB. C. Quantification of percent KS cells with IF signal for LANA-NBs, 3-nitrotyrosine (3-NT) and colocalization of 3-NT with n > 1 LANA-NBs. **p < .01, ***p < .005, Mann-Whitney, unpaired U test. Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

iNOS and nitrotyrosine expression in L1T3 tumor model. A. Western blot of TIVE, L1T3 cells, and L1T3 mouse xenograft tumors assayed for iNOS, LANA, ORF45, and Actin. Molecular weight markers indicated to the left. B. Western blot of TIVE, KSHV-TIVE (KSHV-T), TIVE-BAC16-LANA-WT (BAC16), L1T4 cell lines and L1T3 xenograft tumors 1–4 assayed for iNOS, LANA, and Actin. Tumors 1, 3 and 4 harvested at 1 month. Tumor 2 harvested at 2 months. C. RT-qPCR for two different primer sets of iNOS (NOS2_1, NOS2_2), MMP11, ORF72, LANA, ORF50, PAN gene transcripts in RNA isolated from TIVE, L1T3 cells, and L1T3 tumors taken at 1 month (Tumor 1) or 2 months (Tumor 2). *p < .05, **p < .01, ***p < .001, or ns (not significant), p values determined by two-tailed t-test. D. IHC of TIVE xenograft control (left) or L1T3 tumors (right, enlarged image far right) stained with LANA (top), iNOS (middle) or 3-nitrotyrosine (bottom). E. IF of TIVE xenograft control (top) or L1T3 tumor (bottom) imaged with iNOS (green) or LANA (red), and DAPI (blue)/merge. F. Same as in D, except 3-nitrotyrosine (green). Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

iNOS inhibitor L-NMMA inhibits L1T3 growth in mouse xenograft. A. Mouse xenografts of L1T3 were normalized into groups (n = 10/group) when tumors reached 100 mm³ and treated daily with L-NMMA (25 mg/kg) (red) or vehicle control (black) i. p. B. Tumor volume(top) and body weight (bottom) were measured three times per week for 23 days. ****p < .0001 ANOVA followed by Tukey T-test and multiple comparison test. C. RNA-qPCR analysis tumor samples from mice treated with vehicle or L-NMMA as described in panel A, tested for LANA, ORF75, ORF45 or ORF50 relative to GUSB. ***p < .001, **p < .01, using two-tailed student t-test. D. RNA-seq analysis from tumors samples (n = 2) for both vehicle or L-NMMA treated tumors. Heat map of top significantly changed genes (all pass FDR<5%). E. Top 25 (all pass FDR<5%) significantly affected pathways determined by IPA. F. FDR<5% HALLMARK sets enriched as identified by GSEA. NES = normalized enrichment score. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)