NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-κB signaling

Abstract

Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1-/- mice and a model of urethane-induced tumorigenesis. Nlrx1-/- mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-κB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.

Keywords: NLR; Nod-like receptor; cancer; inflammation; urethane.

Figure 1. NLRX1 Gene Expression is Significantly Dysregulated in Diverse Human Neoplasms

A. A retrospective analysis of gene expression metadata from samples collected from human subjects revealed that NLRX1 expression was significantly dysregulated in a diverse range of cancer subtypes. Data shown were determined to be significant changes in NLRX1 expression between the tumor sample and either adjacent tissue from the same subject or a comparable tissue from an unaffected control subject based on the specific parameters established in each individual study. B-C. Macrophages from Nlrx1^−/− mice rapidly proliferate and release increased cytokines associated with cell growth and migration. B) Bone marrow derived macrophages from Nlrx1^−/− mice significantly expand under standard tissue culture conditions. C) Significant increases in Ccl2 (Monocyte Chemotactic Protein-1) and Csf3 (GCSF) gene expression were observed in Nlrx1^−/− macrophages compared to wild type macrophages. Data are representative of 5 independent studies. *p<0.05.

Figure 2. NLRX1 Improves Survival and Morbidity Following Urethane Exposure

A. Schematic of the urethane model. Nlrx1^−/− and wild type mice received 7 injections of 1g/kg body weight of urethane in saline over the course of 7 weeks. B. Kaplan-Meier plot of Nlrx1^−/− and wild type mouse survival. C. Nlrx1^−/− mice demonstrated a significant decrease in weight gain over the course of the model compared to the wild type animals. D. Necropsy revealed palpable masses in the peritoneal cavity associated with hypersplenomegaly in the Nlrx1^−/− mice. E. Urethane treatment resulted in significant increases in spleen weight in the Nlrx1^−/− mice compared to wild type animals. F. Urethane treatment increases Nlrx1 expression in wild type mice. Data are representative of 3 independent studies. Nlrx1^−/− urethane, n=10; wild type urethane, n=28; Nlrx1^−/− saline, n=3; wild type saline, n=3. *p<0.05.

Figure 3. NLRX1 Attenuates the Development and Progression of Histiocytic Sarcoma

Evaluation of histopathology revealed that urethane treatment resulted in the development of histiocytic sarcoma in Nlrx1^−/− mice. A. Evaluation of H&E stained sections revealed a significant expansion of the white pulp and increased monocyte populations in spleens from urethane treated Nlrx1^−/− mice compared to the saline treated and urethane treated wild type animals. B. Higher magnification evaluation revealed that all of the lesions contained high-grade malignancies consisting of markedly pleomorphic monocytes containing multiple mitotic figures (arrow). C. Semi-quantitative scoring of histopathology revealed a significant increase in spleen lesions in the Nlrx1^−/− mice compared to the wild type animals following urethane treatment. D. Immunohistochemistry using MAC387 (Abcam) was utilized to better classify the pre-dominate cell populations present in the spleen. All of the monocytes associated with spleen lesions were strongly positive for MAC387, suggesting that they are predominately macrophages. Data are representative of 3 independent studies. Nlrx1^−/− urethane, n=10; wild type urethane, n=28; Nlrx1^−/− saline, n=3; wild type saline, n=3. *p<0.05.

Figure 4. NLRX1 Attenuates Urethane Induced Lesions in the Lung and Liver

A. Evaluation of histopathology revealed that urethane exposure increased lung lesions in both Nlrx1^−/− and wild type mice. B. Semi-quantitative scoring revealed a significant increase in bronchial associated lymphoid tissue (BALT) hyperplasia and the number of lesions in lungs harvested from urethane treated Nlrx1^−/− mice. C. In addition to the lungs, urethane exposure also resulted in increased liver lesions in the wild type and Nlrx1^−/− mice. Nlrx1^−/− mice developed significant increases in extramedullary hematopoiesis, perivascular inflammation, and necrosis compared to wild type animals. Data are representative of 3 independent studies. Nlrx1^−/− urethane, n=10; wild type urethane, n=28; Nlrx1^−/− saline, n=3; wild type saline, n=3. *p<0.05.

Figure 5. Genes Associated with Cancer, Autophagy, and Cell Death Are Significantly Up-Regulated in NLRX1 Deficient Mice during Histiocytic Sarcoma

Gene transcription was profiled from RNA collected from wild type and Nlrx1^−/− spleens 14 weeks following the initial saline or urethane exposure. A-B. Heatmap reflecting the change in gene expression of all genes associated with common cancer pathways, autophagy, and cell death that were identified as being significantly up-regulated in the spleen following urethane treatment of Nlrx1^−/− mice compared to the urethane treated wild type animals. Analysis was based on the ΔΔCt method, where all data was standardized to the average gene expression for a panel of 8 housekeeping genes and normalized to the respective untreated Nlrx1^−/− and untreated wild type spleens. Greater than a 2-fold change in gene expression was considered significant. Three - five randomly selected spleens from each genotype and treatment group were selected and pooled for profiling studies. C-D. A significant increase in serum IL-6 and CCL2 levels were detected in the Nlrx1^−/− mice compared to wild type animals 14 weeks after the initial urethane injection. Cytokine levels were determined by ELISA. Nlrx1^−/− urethane, n=6; wild type urethane, n=10; Nlrx1^−/− saline, n=3; wild type saline, n=3. *p<0.05.

Figure 6. NLRX1 Negatively Regulates NF-λB and AKT Signaling during Tumorigenesis

A. Heatmap and fold change in expression of all genes associated with NF-λB signaling that were identified as being significantly up-regulated in the spleen following urethane treatment of Nlrx1^−/− mice compared to the urethane treated wild type animals. Analysis was based on the ΔΔCt method, where all data was standardized to the average gene expression for a panel of 8 housekeeping genes and normalized to the respective untreated Nlrx1^−/− and untreated wild type spleens. Greater than a 2-fold change in gene expression was considered significant. Three – five randomly selected spleens from each genotype and treatment group were selected and pooled for profiling studies. B. Evaluation of all gene expression data using Ingenuity Pathway Analysis revealed a significant increase in NF-λB and AKT signaling in Nlrx1^−/− spleens following urethane exposure compared to wild type. Pink icons represents genes that were significantly up-regulated (>2 fold change in expression) in the Nlrx1^−/− spleens compared to the change in expression observed in the wild type spleens following urethane treatment. Nlrx1^−/− urethane, n=5; wild type urethane, n=5; Nlrx1^−/− saline, n=5; wild type saline, n=5.