Nitric-Oxide Synthase trafficking inducer (NOSTRIN) is an emerging negative regulator of colon cancer progression

Abstract

BackgroundNOSTRIN, abundantly expressed in colon, was reported to be anti-angiogenic, anti-invasive and anti-inflammatory. NOSTRIN expression was inversely related to survival of pancreatic ductal adeno-carcinoma patients. Yet its function and regulatory mechanism in CRC remains elusive.MethodsNOSTRIN’s influence on EMT of CRC cells were analysed using realtime PCR array containing the functional EMT-transcriptome followed by western blotting. Regulation of oncogenic potential of CRC cells by NOSTRIN was elucidated using soft agar colony formation, trans-well invasion, wound healing and colonosphere formation assays. Biochemical assays were used to reveal mechanism of NOSTRIN function. Human CRC tissue array was used to test NOSTRIN mark in control and CRC disease stages.ResultsWe showed here that CRC cell lines with less NOSTRIN expression has more invasive and migratory potential. NOSTRIN affected EMT-associated transcriptome of CRC cells by down regulating 33 genes that were functionally annotated to transcription factors, genes important for cell growth, proliferation, migration, cell adhesion and cytoskeleton regulators in CRC cells. NOSTRIN over-expression significantly reduced soft agar colony formation, wound healing and cell invasion. In line with this, RNA interference of Nostrin enhanced metastatic potential of CRC cells. Furthermore, stable overexpression of NOSTRIN in CRC cell line not only curtailed its ability to form colonosphere but also decreased expression of stemness markers CD133, CD44 and EpCAM. NOSTRIN’s role in inhibiting self-renewal was further confirmed using BrdU incorporation assay. Interestingly, NOSTRIN formed immune-complex with Cdk1 in CRC cells and aided in increase of inhibitory Y15 and T14 phosphorylation of Cdk1 that halts cytokinesis. These ex vivo findings were substantiated using human colon cancer tissue array containing cDNAs from patients’ samples with various stages of disease progression. Significant decrease in NOSTRIN expression was found with initiation and progression of advanced colon cancer disease stages.ConclusionWe illustrate function of a novel molecule, NOSTRIN in curtailing EMT and maintenance of CRC cell stemness. Our data validates importance of NOSTRIN mark during onset and disease progression of CRC indicating its diagnostic potential.