Role of Periostin Expression in Canine Osteosarcoma Biology and Clinical Outcome

Abstract

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.

Keywords: dogs; gene expression; immunohistochemistry; osteosarcoma; periostin.

Figures 1–6.

z-Transformed periostin (POSTN) mRNA expression values in canine osteosarcoma (OS) cell lines (n = 10) as compared to 27 other canine tumor cell lines encompassing a variety of histo-types. Figure 2. Log₂ POSTN mRNA expression in canine primary appendicular OS tumors (n = 49) as compared to normal metaphyseal bone samples (n = 8). Data in Figures 1 and 2 represent mean ± SD, *P < .05 and P < .0001 (unpaired 2-tailed Student’s t-test). Figure 3. Western blot demonstrating antibody detection of a secreted protein of ~ 100 kDa, near the predicted molecular weight (93 kDa) of periostin, in supernatant (lane 3) obtained from the canine Moresco OS tumor cell line. Lane 1 contains protein molecular weight standards and demonstrates the 100 kDa molecular weight marker. Lane 2 consists of Moresco cell line lysate. Figure 4. Immunocytochemistry using this antibody also demonstrated membranous expression of periostin in canine Moresco OS cells. Figures 5 and 6. Immunohistochemical localization of periostin expression in canine formalin-fixed paraffin-embedded positive control (normal) tissues including the tunica media of the aorta (Fig. 5) and colonic mucosa (Fig. 6).

Figures 7–12.

Figures 7 and 8. Osteosarcoma (OS), bone, dog. Representative images demonstrating the variability in positive immunolabeling for periostin in the cytoplasm of neoplastic OS cells from case 346 (Fig. 7) and case 999 (Fig. 8). Figure 9. Quantitative image analysis of positive periostin (POSTN) immunolabeling, expressed as percentage of total tumor area, in 18 OS primary tumors from dogs with a disease-free interval (DFI) <150 days or >250 days. Figure 10. Periostin-positive immunolabeling in dogs with DFI <150 days or >250 days. Mean ± SD. Figure 11. Association between periostin mRNA and protein expression (% positive tumor area) for the same subset of tumors shown in Figure 9. Spearman correlation, r = 0.71, P = .001. Figure 12. Kaplan-Meir survival curve demonstrating the relationship between “low” and “high” periostin mRNA expression in OS primary tumors and disease-free intervals in 49 dogs (log rank P = .59).

Figure 13.

Gene Set Enrichment Analysis (GSEA; Hallmark gene sets of Molecular Signatures Database) of differentially enriched pathways in canine osteosarcoma primary tumors with high (n = 24) versus low (n = 25) periostin mRNA expression. Hallmark gene sets ranked according to normalized enrichment score (NES) and false discovery rate (FDR)-adjusted q value, *q < 0.05, **q < 0.01, ***q < 0.001, and ****q < 0.0001.

Figures 14–16.

GSEA enrichment plots of specific gene sets upregulated in periostin “high” versus periostin “low” canine OS primary tumors.

Figures 17–19.

GSEA enrichment plots of specific gene sets upregulated in periostin “low” versus periostin “high” canine OS primary tumors.

Figure 20.

Relative expression of immune cell lineage markers in high (n = 12) and low (n = 12) periostin-expressing tumors. Heatmap colors represent z-transformed expression values. Color bars on the top and side specify the periostin expression and immune cell type, respectively. Disease-free intervals (DFI) are represented in the histogram. Asterisks denote markers differentially expressed between high versus low periostin-expressing tumors (FDR q < 0.05 and fold-change of at least 1.5).

Figure 21.

Immune cell scores between high and low periostin-expressing samples. Statistical significance was determined using multiple unpaired t tests with Benjamini, Krieger, and Yekutieli’s correction for multiple comparisons (q < 0.05).