DISCUSSION
COX-2 has been described as a critical element in the crosstalk between cancer and inflammation in NB and other solid tumors18. However, the mechanisms involving COX-2 in tumorigenesis and the therapeutic significance of COX-2 inhibition remain elusive, especially in pediatric tumors 16, 30, 36, 37. It is not clear, for instance, if leukotriene inhibitors would be safe and effective as adjuvant therapy, at what phase of treatment, or what molecular interactions could be affected by its inhibition. In the present work, we show that COX-2 was expressed in all the analyzed NB samples, similar to previous findings 38. However, COX-2 expression levels were significantly higher in post-CT specimens when compared with samples collected before treatment. The clinical validation in a higher NB patient cohort would support non-steroidal anti-inflammatory drugs in the consolidation or maintenance of NB chemotherapy regimens. By combining clinical and genomic data with network topological analysis, we also show that higher COX-2 expression may arise from the loss of a complex dynamic of interactions between components of the inflammation pathways and protein-coding genes located on Ch11q.
MNA and Ch11q LOH are independent poor prognostic factors of high-risk NB9, almost mutually exclusive, with similar significance for prognosis 10. Ch11q LOH and NAMN were observed in three patients of this study (Group 2), presenting paired specimens, either in the pre- or post-CT tumors. Expectedly, these patients presented poor prognosis factors, such as advanced age at diagnosis and a high number of CNAs. Group 2 also showed a higher expression of COX-2 protein when compared with the other groups, regardless of the MYCN status. To comprehend the correlation of Ch11q deletions and COX-2 expression, we built a PPI network including the genes located in this deleted region, COX-2/PGE2 pathway genes, and the target genes of the miRNAs codified in the deleted 11q regions. Clustering and GO analysis identified five clusters, with genes involved in different biological processes, primarily related to cancer hallmarks39. Processes, such as regulation of apoptosis, cell proliferation, and inflammation, and the ones associated with collagen metabolism were observed in cluster 1. Cluster 3 was associated with the prostaglandin metabolic process, while cluster 5 showed an extensive network of neurological system regulation and IL-6 signaling as a particular inflammation pathway.
The nodes classified as HBS can be considered the ones with stronger topological influence, which can be due to their multiple direct interacting partners and their control of the network information flow. The genes IL-1β , CEBPB , EP300 , and STAT3,were identified as the HBS nodes acting as integration bridges among clusters of interest. Noteworthy, all these genes were inputs from the inflammation pathways. Although molecular connections among such elements have not been shown for NB yet, other biological models demonstrate their correlation (Supplemental Figure S3). CEBPB andEP300 genes that codify for the C/EBPβ and p300 proteins, respectively, are both activators of PTGS2 transcription40, 41. IL-1β protein, generated predominantly by tumor-infiltrating macrophages within the tumor microenvironment (TME), has a more functional regulatory role in COX-2 activation, regulating the expression of MMPs 42, as reported for breast43 and colorectal cancers 44.
MMPs are zinc-containing endopeptidases that act on the extracellular matrix (ECM). These proteins are overexpressed in CAFs and generate ECM cleavage products, such as fibronectin and collagen, which serve as chemotactic factors for inflammatory cells 45.MMP-3 is a direct transcriptional target and an essential contributor to the Wnt/β-catenin signaling pathway 46. In our PPI network, MMP3 was an HBS associated with Cluster 1. It directly interacts with other MMPs and also with CTNNB1 , which in turn, is linked to inflammation related HBS (EP300 , TNF,and JUN ). The functions of MMP-3 are linked toMMP-1 , MMP-7 , MMP-10 , and MMP-13 , all identified in the constructed PPI network, are involved in collagen catabolic and metabolic processes 47 that ultimately determine the differentiation of tumor-associated immune cells48.
In summary, the PPI network analysis shed light on the conjugation of the metalloproteases with the inflammation-related genes in an indirect mode of interaction with PTGS2 . We suggest that the deficiency in MMPs, which are essential for accurate remodeling of collagen in Ch11q deleted tumors, may generate a pro-inflammatory signaling profile involving increased activities of COX-2 and other enzymes from the PGE2 production pathway through IL-1β activation. It is essential to mention that the loss of Ch11q does not occur alone but in association with other chromosomal aberrations in NBs, such as the loss of chromosomes 3p and 4p and gain on chromosome 7q 49; however, further studies are required due to its the association with chemoresistance. Along the same line, miRNA targets found in the Ch11q region of interest were included to build our network. Interestingly 33% of these miRNA targets are coded by chromosome 1
Although the sample size limitation, the present biological analysis of network systems strategy allowed the analysis of the interaction of multiple molecular pathways, providing novel and complementary biological insights to the NBs tumorigenesis. Once these findings are clinically validated in larger and independent well-annotated cohorts of NBs patients and experimental cellular models, these findings might be translated into clinical applications for the prognosis and treatment of patients with NB.
DISCLOSURE STATEMENT: None Conflict of Interest