Elevated TNFR1 and serotonin in bone metastasis are correlated with poor survival following bone metastasis diagnosis for both carcinoma and sarcoma primary tumors

Abstract

Purpose: There is an urgent need for therapies that will reduce the mortality of patients with bone metastasis. In this study, we profiled the protein signal pathway networks of the human bone metastasis microenvironment. The goal was to identify sets of interacting proteins that correlate with survival time following the first diagnosis of bone metastasis.

Experimental design: Using Reverse Phase Protein Microarray technology, we measured the expression of 88 end points in the bone microenvironment of 159 bone metastasis tissue samples derived from patients with primary carcinomas and sarcomas.

Results: Metastases originating from different primary tumors showed similar levels of cell signaling across tissue types for the majority of proteins analyzed, suggesting that the bone microenvironment strongly influences the metastatic tumor signaling profiles. In a training set (72 samples), TNF receptor 1, alone (P = 0.0013) or combined with serotonin (P = 0.0004), TNFα (P = 0.0214), and RANK (P = 0.0226), was associated with poor survival, regardless of the primary tumor of origin. Results were confirmed by (i) analysis of an independent validation set (71 samples) and (ii) independent bioinformatic analysis using a support vector machine learning model. Spearman rho analysis revealed a highly significant number of interactions intersecting with ERα S118, serotonin, TNFα, RANKL, and matrix metalloproteinase in the bone metastasis signaling network, regardless of the primary tumor. The interaction network pattern was significantly different in the short versus long survivors.

Conclusions: TNF receptor 1 and neuroendocrine-regulated protein signal pathways seem to play an important role in bone metastasis and may constitute a novel drug-targetable mechanism of seed-soil cross talk in bone metastasis.

Figure 1. Bone metastasis molecular network in short-and long-survivors

Interacting proteins comprising the bone metastasis molecular network are strikingly different in short-survivors (survival ≤ 5 months) compared to-long-survivors (survival ≥ 38 months), using Spearman’s ρ correlation analysis data for the sample set in this study. Correlations with ρ≥0.75 and p≤0.01 were used to build the network graph (Gephi 0.8.1 beta, The Gephi Consortium, Paris, France, www.gephi.org). Each node represents a molecule: the bigger the node, the more the significant correlations relative to the molecule. Each line connecting 2 nodes represents a significant correlation between the nodes: the thicker the line, the higher the Spearman’s ρ correlation. Proteins are grouped base on Spearman’s ρ values and the number of connections among a group of nodes: strongly correlated nodes are represented closed to each other and with the same color. In short-survivors, the molecular network is dominated by a group of interconnections between TNFα, RANKL, ERα S118 and Serotonin. Interconnections between nodes are wider and less numerous in long-term survivors and lack a dominant network.

Figure 2. TNFR1 and bone regulating proteins correlate with patient’s survival in the training set

Graphs representing Kaplan-Meier survival curves for TNFR1 and combination of TNFR1 with one or more proteins regulating bone metabolism in the training set. The median of the expressions of the end-point (or combination of end-points) was used as the cut point to create the two groups, high expressing patients (high) and low expressing patients (low). Combination of end-points was achieved by summing together the end-point expression values. Survival was defined as the number of months each patient survived following the first diagnosis of bone metastasis.

Figure 3. Correlation of TNFR1 and bone regulating proteins with patient’s survival is validated in an independent set

Graphs represent Kaplan-Meier survival curves for TNFR1 and combination of TNFR1 with one or more proteins regulating bone metabolism in the validation set. The median of the expressions of the end-point (or combination of end-points) was used as the cut-point to create the two groups, high expressing patients (high) and low expressing patients (low). Combination of endpoints was achieved by adding together the end-point expression values. Survival was defined as the number of months each patient survived following the first diagnosis of bone metastasis.

Figure 4. TNFR1 expression inversely correlates with survival over the full range of the survival curve over all 143 samples including all primary tumor groups

In all four graphs survival is defined as the number of months each patient survived following the first diagnosis of bone metastasis. A) The total set of 143 samples with known survival data was subdivided into 4 quartiles base on survival time. The mean and median TNFR1 expression in the 4 quartiles descends over the shortest to the longest survivors. The Mann-Whitney test was applied to medians of the lowest (x≤5) and highest (x≥38) survival quartiles. The Kruskal-Wallis test was performed on the medians of 4 groups of samples. B) This graph depicts the Kaplan-Meier survival curves in the total set of 143 samples, using the median of TNFR1 expressions as the cut point to create the high expressing (high) and the low expressing (low) patients’ groups. C) Kaplan-Meier survival curves for the 25^thand 75^th quartile of TNFR1 expression in the 143 sample set. D) Kaplan-Meier curves showing the survival distribution in each primary tumor origin group of patients (color lines) and in the total set of 143 samples (black line).