# ErbB4 Is a Potential Key Regulator of the Pathways Activated by NTRK-Fusions in Thyroid Cancer
## 3 Results
### Regulators of Uregulated Genes Associated with the Nervous System  
Returning to the fact that genes associated with nervous system development were upregulated only in the thyroid cancer samples with _NTRK1_ or _NTRK3_ gene fusions (Figure 5), we specifically searched for upstream regulators for these genes. In total, we identified 29 upregulated genes that were related to the following GO categories associated with the nervous system (e.g., \"axon\", \"nervous system process\", \"synapse assembly\", \"axonogenesis\") (Supplementary Table S3). Among them, the highest gene expression difference between the samples with and without NTRK-fusions was for the genes: _AUITS2_, _DTNA_, _ERBB4_, _FLRT2_, _FLKT3_, _RPH3A_, and _SCN4A_.  
Figure 3: The log2 transformed foldchange values for genes encoding the upstream regulators identified. Samples with _NTRK1_ gene fusions versus samples without NTRK fusions (**A**) and samples with _NTRK3_ fusions versus samples without NTRK fusions (**B**). Genes with maximally different log2 FoldChange values are annotated with arrows and gene symbols.  
We suggest that the activation of these genes is a consequence of NTRK-fusion occurring in thyroid tumor tissue and the subsequent rewiring of the key signaling pathways in these tumors towards the further activation of their growth. Such potential rewiring in the network can be identified with the help of the search for positive feedback loops, \"walking pathways\" [18], that is implemented in the Genome Enhancer tool. Therefore, we applied the Genome Enhancer tool to analyze the 29 genes identified to find key master regulators that may enhance their expression in the studied tumor types. During the first step, Genome Enhancer analyzed the promoters of the 29 upregulated genes, applying the F-Match [19] and CMA [24] tools using the TRANSFAC motif library [25]. As a result, clusters of binding sites were revealed for the following transcription factors: NFkB, Oct-1, Egr2, and KLF4 in the promoters of these up-regulated genes (Supplementary Figure S1), explaining the details of the molecular mechanism of their transcriptional control in tumors of the studied types. Next, Genome Enhancer, with the help of the algorithm \"walking pathways\" [18], used the database TRANSPATH [23] and performed a search in the signal transduction network upstream of the revealed transcription factors, and revealed four key master regulators (encoded by the genes _ERBB4_, _DKK1_, _NOG_, and _ARHGEF7_). ErbB4 was determined as the top key master regulator. Finally, we reconstructed the potential regulatory network connecting activated ErbB4 and a few other key master-regulators molecules with the identified upregulated nervous system development genes in the thyroid tumors carrying _NTRK1_- and _NTRK3_-fusions (Figure 6).  
Figure 4: The comparison of the normalized gene expression value distributions for the genes regulated by _EGFR_ (**A**) and _ERBB4_ (**B**) in normal thyroid gland tissues (first boxes), thyroid cancer without NTRK gene fusion (second boxes), with _NTRK1_ (third boxes) and _NTRK3_ (fourth boxes) gene fusions.  
Using the STRING database, we constructed an interaction network for the top up-regulated genes and associated master regulators identified in our analysis (Figure 7). For proteins encoded by _ERBB4_, _EGFR_, _BMX_, _DKK1_, _NOG_, _ARHGEF7_, and _NTRK1_, various interactions were documented in the STRING database--experimentally determined physical interactions and co-expression of their homologs in other organisms, or relations between genes found by text mining of scientific publications. Some of these interactions may play an essential role during thyroid cancer development.