Nod2 protects from orthotopic 4T1 breast tumor
To test our hypothesis that Nod2 protects mice from the development of breast tumors, we injected 4T1 cells into the abdominal mammary fat pad of WT and Nod2−/− female mice. 4T1 is a mammary carcinoma cell line that rapidly establishes tumors when injected into the mammary tissue of mice, and thus provides a well-established orthotopic breast cancer model [30, 31]. Tumors were visible by day 3 after the injection and on day 6 mice were sacrificed and tumors were extracted, measured, and analyzed. Nod2−/− mice developed larger tumors; the tumor weight and volume were significantly higher in Nod2−/− mice than in WT mice (Fig. 1A, B, and C). There was also a significant difference between the two groups in the percentage of mice that developed tumors; 100% of Nod2−/− mice developed tumors, whereas only 50% of WT mice developed tumors (Fig. 1D). These results indicate that Nod2 protects mice from the development of the transplanted 4T1 mammary carcinoma.
Development of breast tumors in Nod2−/− mice is associated with differential activation of lipid metabolism, cell cycle, and cancer pathways
To unravel the molecular mechanism underlying the development of breast tumors in Nod2−/− mice in an unbiased manner, we performed RNAseq using total RNA from the tumors of WT and Nod2−/− mice injected with 4T1 cells. Compared with WT mice, Nod2−/− mice had significantly altered expression of over 3,000 genes with at least 5% FDR (false discovery rate, P ≤ 0.05). Of these 3,000 genes, over 800 genes had ≥2-fold increase in expression and over 2,000 genes had ≤2-fold decrease in expression in Nod2−/− mice (Supplementary Fig. S1). We analyzed the differentially expressed genes using Ingenuity Pathway Analysis software (Qiagen) to identify Gene Ontology (GO) biological functions and diseases that are altered between WT and Nod2−/− mice [24]. Based on the number of genes, the most altered biological functions between WT and Nod2−/− mice were lipid metabolism and cell cycle (Table 1). The top three diseases that were significantly altered between WT and Nod2−/− mice were cancer, endocrine system disorders, and reproductive system diseases (Table 1).
Table 1
Top Gene Ontology biological functions and diseases significantly altered in the tumors of Nod2−/− mice compared with WT mice.
Gene Ontology Biological Functions and Diseases | Number of genes | P-value range |
Biological Function | Lipid Metabolism | 405 | 1.51E-07 to 1.23E-30 |
Cell Cycle | 345 | 1.42E-07 to 6.22E-24 |
Disease | Cancer | 1584 | 1.52E-07 to 1.70E-37 |
Endocrine System Disorders | 1348 | 1.67E-07 to 1.80E-32 |
Reproductive System Diseases | 1080 | 1.52E-07 to 9.27E-29 |
We further analyzed our transcriptomics data for specific pathways associated with the biological functions and diseases that are significantly altered between WT and Nod2−/− mice (Table 2). Nod2−/− mice had a significantly increased activation of DNA replication, DNA repair, cell cycle and its regulation (Table 2). By contrast, Nod2−/− mice had a significantly decreased activation of biosynthesis and hydrolysis pathways for fatty acids, triglycerides, and cholesterol (Table 2). Nod2−/− mice also had a decreased activation of adipogenesis. We analyzed our transcriptomics data for differential activation of regulatory molecules and signaling pathways between WT and Nod2−/− mice. The PPARα/PPARγ and LXR/RXR signaling pathways were significantly inhibited, whereas the ErbB signaling pathway was significantly activated in Nod2−/− mice. The glutathione-mediated detoxification and Nrf-2-mediated oxidative stress response were also inhibited in Nod2−/− mice compared with WT mice.
We next identified individual genes associated with these pathways with a focus on genes that have ≥2-fold increase or decrease in expression and 5% or lower FDR between the two groups of mice. The change in expression (fold change) of individual genes in Nod2−/− mice compared with WT mice is shown as heat maps and the genes are grouped together by their function/pathway. Many genes belong to multiple pathways, but in the heat maps, genes are only included in their identified primary pathway.
Table 2. Top Gene Ontology pathways that are significantly altered in the tumors of Nod2 −/− mice compared with WT mice.
Gene Ontology Pathways | P-value |
DNA replication and repair | DNA replication | 5.012E-11 |
Deoxyribonucleotide de novo biosynthesis | 6.760E-3 |
Nucleotide excision repair | 2.911E-02 |
Mismatch repair | 8.511E-04 |
Double-strand DNA repair | 3.467E-05 |
Cell cycle and regulation | Cyclins and cell cycle regulation | 4.074E-4 |
Checkpoint regulation | 1.950E-05 |
Lipogenesis | Fatty acid biosynthesis | 1.778E-05 |
Triglyceride biosynthesis | 1.862E-06 |
Phosphatidylglycerol biosynthesis | 7.244E-04 |
Lipolysis | Mitochondrial fatty acid oxidation | 1.995E-16 |
Peroxisomal beta-oxidation | 1.738E-03 |
Steroid biosynthesis | Cholesterol biosynthesis | 2.630E-05 |
Estrogen biosynthesis | 3.467E-03 |
TCA cycle & pyruvate metabolism | TCA cycle | 1.738E-3 |
Biosynthesis of acetyl CoA | 1.023E-3 |
Adipocyte biology | Adipogenesis | 1.445E-04 |
Signaling | PPARα and PPARγ | 1.810E-36 |
LXR/RXR | 9.550E-4 |
ErbB | 5.010E-60 |
Stress response | Glutathione-mediated detoxification | 3.162E-08 |
Nrf2-mediated oxidative stress response | 4.786E-03 |
Development of breast tumors in Nod2−/− mice is associated with increased expression of genes involved in DNA replication and repair, and in cell cycle
Based on our transcriptomic data, Nod2−/− mice had a significantly higher activation of DNA replication compared with WT mice (Table 2). Genes that code for proteins required for replication were overwhelmingly upregulated in the tumors of Nod2−/− mice and included enzymes and accessory proteins: DNA polymerases (Pola1, Pola2, Pole), primases (Prim1, Prim2), topoisomerases (Top2a, Topbp1), and DNA helicase (minichromosome maintenance complex, Mcm2-7, Mcm10) (Fig. 2 and Supplementary Table S1). Genes coding for proteins that mediate chromatin assembly and integrity (Chaf1a, Chaf1b, Dnmt1, Hat1, Uhrf1) were also upregulated. The expression of genes that code for proteins involved in nucleotide synthesis or salvage pathways (Cdadc1, Ctps, Dck, Mtap, Rrm2, Tyms, Uck2) was also significantly increased in Nod2−/− mice.
Nod2 −/− mice had an increased activation of DNA repair pathways, including nucleotide excision repair, mismatch repair and double-stranded DNA repair pathways, compared with WT mice (Table 2). Genes in nucleotide excision repair (Ercc1, Rad18), mismatch repair (Exo1, Msh2, Msh6), double strand DNA repair (Blm, Brca1, Brca2, Fancd2, Mre11a, Rad21), and genes that participate in more than one repair pathway (Dna2, Fen1, Pole2, Rad51, Rad51c, Rad54b, Rfc3) were upregulated in the tumors of Nod2−/− mice compared with WT mice (Fig. 2 and Supplementary Table S1).
Nod2 −/− mice had a significantly higher activation of cell cycle, which includes cyclins and cell cycle regulation and checkpoint regulation compared with WT mice (Table 2). Genes that code for proteins required for or promoting the cell cycle were overwhelmingly upregulated in Nod2−/− mice and included genes that code for cyclins (Ccna2, Ccnb2, Ccnd1, Ccnf), cyclin-dependent kinases and accessory proteins (Cdk1, Cdk2, Cdk6, Cks1b, Cks2), Aurora kinases (Aurka, Aurkb), and E2F transcription factors (Cdca4, E2f1, E2f7, Edf8). Nod2−/− mice had significantly higher expression of genes that code for centromere proteins that are essential for forming the kinetochore or attachment of the chromosomes to the mitotic spindle (Cenpa, Cenpc1, Cenpe, Cenpf, Cenph, Cenpi, Cenpl, Cenpn, Cenpt, Cenpu, Incenp) and for several cell cycle associated ubiquitin ligases and accessory proteins required for ubiquitination (Cdc20, Dtl, Fbxo5, Skp2, Ube2s). Nod2−/− mice had significantly higher expression of the cell proliferation markers (Mki67, Mybl2, Myc) compared with WT mice (Fig. 2 and Supplementary Table S1). By contrast, there was decreased expression of several cell cycle inhibitors (Cdkn1c, Cdkn2b, Cdkn2c, Cpped1, Hdac11, Inca1, and Tspyl2).
These results demonstrate that the tumors in Nod2−/− mice have increased activation of genes that promote DNA replication and cell division, which likely contributes to the higher incidence and larger 4T1 tumors in Nod2−/− mice compared with WT mice.
Development of breast tumors in Nod2−/− mice is associated with decreased expression of genes involved in lipid and steroid metabolism and signaling
Based on our transcriptomic data, lipid metabolism pathways, both biosynthesis and hydrolysis, were overwhelmingly downregulated in Nod2−/− mice compared with WT mice (Table 2). Nod2−/− mice had significantly decreased expression of genes coding for enzymes in fatty acid synthesis (Acaca, Acacb, Acly, Acot1, Acot2, Acot4, Acot7, Acss2, Fasn) and desaturation of fatty acids (Cyb5a, Cyp4b3, Fads3, Fads6, Scd1, Scd2, Scd3, Scd4) (Fig. 3 and Supplementary Table S2). Genes coding for enzymes in phospholipid and triacylglycerol synthesis (Agpat2, Agpat3, Chpt1, Dgat1, Dgat2, Gpam, Gpat3, Gpat4, Lpcat3, Lpin1, Mogat1, Ormdl3, Oxct1, Plpp2, Plpp3, Ptdss2), and its regulators (Me1, Midlip1, Mlxipl) were downregulated. The expression of genes coding for enzymes in choline metabolism and its transport (Chdh, Slc44a4), citrate transporter (Slc25a1), and ketone body metabolism (Acat1, Acss3, Hmgcs2, Oxct1) was also significantly decreased in Nod2−/− mice (Fig. 3 and Supplementary Table S2).
Nod2 −/− mice also had a significantly decreased expression of genes involved in fatty acid oxidation and triglyceride hydrolysis. Genes coding for carnitine/acyl-carnitine transporters (Cpt2, Crat, Slc22a4, Slc22a5) and lipid transporters (Abca6, Abca9, Abcd2, Cd36) were downregulated in Nod2−/− mice compared with WT mice (Fig. 3 and Supplementary Table S2). Nod2−/− mice had significantly decreased expression of genes coding for enzymes in mitochondrial fatty acid oxidation (Acaa2, Acad10, Acadm, Acads, Acadsb, Acadvl, Acsl1, Ecsh1, Hadh, Hadha, Hadhb) and peroxisomal fatty acid oxidation (Abcd2, Acaa1a, Acaa1b, Acox1, Ech1, Eci3, Hsd17b4). Genes coding for enzymes and their regulators in triglyceride hydrolysis (Abhd5, Lipe, Lpl, Mgll, Pnpla2, Pnpla3) and oxidation of odd-chain fatty acids (Mcee, Mut, Pcca, Pccb) were also downregulated in Nod2−/− mice (Fig. 3 and Supplementary Table S2).
Nod2 −/− mice had a significantly decreased activation of steroid biosynthesis, including estrogen biosynthesis, compared with WT mice (Table 2). Genes coding for proteins in cholesterol synthesis (Acat2, Cyp2d22, Cyp2e1, Cyp2f2, Cyp2j9, Nsdhl) and cholesterol transport (Abca2, Abca5, Abca8a, Gramd1b, Scp2, Stard4) were downregulated in Nod2−/− mice (Fig. 3 and Supplementary Table S2). The expression of genes for enzymes involved in the synthesis and activation of sex hormones (Akr1c14, Akr1c18, Gsta3, Hasd17b8, Hsd17b12, Hsd17b14) and signaling by these hormones (Esr, Esrra, Ncoa1, Osbpl1a, Pgr, Rxra, Wbp2) was also downregulated in Nod2−/− mice.
The LXR/RXR signaling pathway was also downregulated in Nod2−/− mice compared with WT mice (Table 2). LXRs are nuclear receptors that form heterodimers with RXR, bind oxysterols, and are important regulators of cholesterol, fatty acid, and glucose homeostasis. Genes that were significantly downregulated in Nod2−/− mice and regulated by LXR include many genes in lipid metabolism (Acaca, Cd36, Echs1, Fasn, Hadh, Lpl, Osbpl1a, Scd1, Scd3, Srebf1). SREBF1 is a transcription factor with a critical role in promoting the expression of genes in lipid metabolism. The lower expression of Srebf1 in Nod2−/− mice correlates with decreased lipid metabolism in these mice. Expression of Por, which codes for cytochrome P450 oxidoreductase, an enzyme required for the activity of cytochrome P450 enzymes, was also significantly downregulated in Nod2−/− mice compared with WT mice (Fig. 3 and Supplementary Table S2).
In addition to lipid metabolism, the TCA cycle and biosynthesis of acetyl CoA were significantly inhibited in the tumors of Nod2−/− mice compared with WT mice (Table 2). Nod2−/− mice had significantly decreased expression of genes that code for enzymes in the TCA cycle (Aco1, Aco2, Cs, Mdh1, Ogdh, Sdha) and proteins in pyruvate metabolism and transport (Pcx, Pdha1, Pdhb, Pdhx, Pdk2, Pdk3, Pdp2, Slc16a3). The transport of pyruvate into the mitochondria and its conversion to acetyl CoA are critical for the TCA cycle and for the fatty acid biosynthesis. Thus, decreased transport and metabolism of pyruvate would result in decreased fatty acid synthesis, and expression of genes involved in both these pathways was lower in Nod2−/− mice compared with WT mice.
Our results demonstrate that the tumors in Nod2−/− mice have significantly decreased expression of genes involved in both lipolysis and lipogenesis compared with the tumors in WT mice. Nod2−/− mice also have decreased expression of genes involved in steroid metabolism and signaling.
Development of breast tumors in Nod2−/− mice is associated with decreased expression of genes involved in adipogenesis and PPAR signaling
Based on the analyses of our transcriptomic data, adipogenesis was significantly inhibited in the tumors of Nod2−/− mice compared with WT mice (Table 2). Nod2−/− mice had significantly decreased expression of genes involved in the formation of lipid droplets (Bscl2, Btn1a1, Ccdc3, Cidec, Fitm2, Methig1, Mettl7a2, Plin1, Plin4, Plin5) and adipocyte metabolism (Aacs, Adrb3, Angptl8, Fabp4, Ffar4). Genes for proteins that promote adipocyte differentiation (Aamdc, Adig, Angptl8, Apmap, Arxes1, Atf5, Bmp4, Cebpa, Cmkir1, Fzd4, Lgals12, Nsd2, Pparg) were also downregulated in Nod2−/− mice (Fig. 4A and Supplementary Table S3).
Nod2 −/− mice had many changes in the expression of genes involved in adipocyte signaling and overall, these changes would result in inhibition of adipogenesis or increased adipocyte dedifferentiation. There was increased expression of genes for the adipokine apelin and its receptor (Apln, Aplnr), an inhibitor of adipogenesis. In contrast, the expression of genes for the adipokine adiponectin (Adipoq) and its receptor (Adipor2), which regulate metabolism in adipocytes and promote adipogenesis, was significantly decreased in Nod2−/− mice. Insulin has a critical role in promoting adipocyte differentiation and genes that code for components of the insulin signaling pathway were downregulated (Akt2, Fcor, Foxo1, Foxo4, Irs1), whereas expression of Fhl2, an inhibitor of Foxo1, was upregulated (Fig. 4A and Supplementary Table S3). The expression of fibroblast growth factor 10 (Fgf10), which stimulates preadipocyte proliferation and adipogenesis, was also downregulated in Nod2−/− mice. The expression of Wnt receptor frizzled 6 (Fzd6), which is often increased in triple-negative breast cancer, was upregulated in Nod2−/− tumors compared with WT tumors. The expression of bone morphogenetic protein 4 (Bmp4), a member of the TGFβ family that promotes adipocyte maturation and differentiation, was decreased in Nod2−/− mice. The expression of Gli3, a transcription factor activated by Hedgehog signaling, which suppresses adipogenesis, was upregulated in Nod2−/− mice (Fig. 4A and Supplementary Table S3).
We also analyzed our transcriptomics data for regulatory molecules that are differentially activated between WT and Nod2−/− mice. Based on the P value, the top 5 regulators included PPARα and PPARγ, which are nuclear receptors that form heterodimers with RXR protein and regulate transcription of genes involved in lipid and carbohydrate metabolism and cell proliferation and differentiation. PPARγ is expressed predominantly in the adipose tissue and induces expression of many genes involved in adipogenesis, including Acox1, Acsl1, Ascl4, Cebpa, Cd36, Cpt2, Fabp4, Lpl, Plin1, Scd1, Scd4, Slc27a1, Slc27a4, which were differentially regulated between WT and Nod2−/− mice. The transcription factor CEBPα also plays a critical role in promoting adipocyte growth and differentiation. The expression of CEBPα and its activator ATF5 were both significantly decreased in Nod2−/− mice. The expression of Foxc2, a transcription factor associated with epithelial to mesenchymal transition, was upregulated in Nod2−/− mice (Fig. 4 and Supplementary Table S3). Our STRING analysis demonstrates a central role for PPARγ in regulating genes involved in adipogenesis (Fig. 4B). Accordingly, Nod2−/− mice had significantly lower levels of total triglyceride and cholesterol compared with WT mice (Fig. 4C).
Our results demonstrate that the development of 4T1 tumors in Nod2−/− mice is associated with a dramatic decrease in the expression of genes involved in lipid metabolism and adipogenesis, which was accompanied by increased delipidation.
Development of breast tumors in Nod2−/− mice is associated with decreased expression of stress-response genes and increased ErbB signaling
Nod2 −/− mice were predicted to have significantly decreased activation of glutathione-mediated detoxification and Nrf2-mediated oxidative stress response pathways compared with WT mice (Table 2). Nrf2 is a transcription factor that induces the expression of genes involved in detoxification reactions. Nod2−/− mice had significantly decreased expression of genes for glutathione peroxidase (Gpx3, Gpx4), members of the glutathione S-transferase family (Gsta3, Gsta4, Gstk1, Gstm1, Gstm2, Gstm3, Gstm4, Gstm5), microsomal glutathione S-transferase (Mgst1, Mgst2, Mgst3), members of the aldehyde dehydrogenase family (Aldh1l1, Aldh1l2, Aldh2, Aldh3b2, Aldh3b3, Aldh4a1), and heat shock proteins (Dnajb2, Dnajc4, Dnajc15) (Fig. 5A and Supplementary Table S4).
Based on the analyses of our transcriptomic data, Nod2−/− mice were predicted to have significantly increased activation of the ErbB signaling pathway compared with WT mice (Table 2). The ErbB receptor tyrosine kinase family consists of four cell surface receptors, ErbB1, ErbB2, ErbB3, and ErbB4, and plays a critical role in many cancers. There was no significant difference in the expression of ErbB1, ErbB2, and ErbB4 between Nod2−/− and WT mice, whereas the expression of ErbB3 was significantly decreased in Nod2−/− mice (Fig. 5 and Supplementary Table S4). However, Nod2−/− mice had significantly increased expression of ligands that bind and activate ErbB receptors (Epgn, Ereg, Hbegf, Nrg1, Tgfa) and downstream targets, including phosphatases that inhibit the MAP kinase pathway (Dusp5, Dusp6) and integrin subunits (Itga2, Itga3, Itga5, Itgb1) (Fig. 5A and Supplementary Table S4).
The role of Nod2 in innate immunity and inflammation is well characterized. A deficiency in Nod2 is linked to increased inflammation and the development of Crohn’s disease and colorectal cancer and may also contribute to the development of obesity and hepatocellular carcinoma [7, 8, 21–24]. However, in our breast cancer model, Nod2−/− mice did not develop a strong immune response. Therefore, inflammation likely does not play a big role in the increased sensitivity to tumorigenesis in Nod2−/− mice compared with WT mice. There were few immune response genes differentially expressed between WT and Nod2−/− mice. Nod2−/− mice had significantly higher expression of the chemokine genes, Ccl3, Cxcl1, Cxcl3, Cxcl10 and Cxcl11, which suggests that there may be increased neutrophil and macrophage infiltration in the tumors of these mice. Nod2−/− mice also had higher expression of aconitate decarboxylase (Acod1) gene, which converts cis-aconitate to itaconate, a regulator of innate immunity and metabolism (Fig. 5A and Supplementary Table S4).
We further identified differences in the expression of keratins and collagens between WT and Nod2−/− mice. Keratins (cytokeratins) are the major cytoskeletal proteins in epithelial cells and include ~ 20 different proteins, whereas collagens are the predominant proteins in the extracellular matrix and include ~ 28 different proteins. The expression of individual keratins and collagens is frequently altered in cancer and specific changes help differentiate between subtypes of a cancer. The tumors in Nod2−/− mice had significantly higher expression of genes for several keratins, including Krt1, Krt6a, Krt6b, Krt10, Krt15, Krt16, several collagens, including Col1a1, Col6a3, Col8a1, and Col12a1, and inhibitors of proteases (Serpine1, Serpine2) that modify the extracellular matrix (Fig. 5A and Supplementary Table S4).
We next analyzed the activation of downstream proteins, STAT3, STAT5, and ERK1/2, which are activated by multiple signaling pathways, including ErbB, and are often activated in many cancers. Nod2−/− mice had significantly higher levels of p-STAT3, lower levels of p-STAT5 and no difference in levels of p-ERK1/2 compared with WT mice (Fig. 5B and C). These results correlate with the decreased expression of Stat5a (Fig. 3) and the increased expression of MAPK inhibitors (Dusp5, Dusp6) in Nod2−/− mice (Fig. 5A).
Development of breast tumors in Nod2−/− mice is associated with altered expression of genes associated with cancer
We next determined whether the genes in the biological pathways differentially activated in Nod2−/− mice (Table 1 and Figs. 2–5) have been previously identified in cancer patients or in cancer models. The vast majority of the differentially expressed genes between WT and Nod2−/− mice have been previously identified either in cancer patients, including breast cancer, or in cancer models (Table 3). These genes are marked with an asterisk in the heat maps (Figs. 2, 3, 4, and 5).
Table 3
Genes that are differentially expressed in tumors of Nod2−/− mice compared with WT mice are associated with cancer.
Pathways | Number of genes with a 2-fold change (Nod2−/−/WT), P≤0.05 | % Genes associated with cancer |
DNA replication and repair | 80 | 100 |
Cell cycle | 105 | 97 |
Lipolysis | 53 | 91 |
Lipogenesis | 55 | 84 |
Steroid biosynthesis & signaling | 45 | 91 |
Pyruvate metabolism & TCA cycle | 18 | 100 |
Adipogenesis | 49 | 73 |
ErbB signaling | 28 | 96 |
Stress response | 34 | 88 |
Immune response | 17 | 94 |
Keratins | 9 | 100 |
Collagen and protease inhibitors | 10 | 100 |