Novel Role of Nitric‐Oxide Synthase Trafficking Inducer (NOSTRIN) in Combating Colon Cancer Aggressiveness

Abstract

Colorectal cancer (CRC) is a global health problem, with approximately 1.2 million new cases per year. CRC is the third most common cancer in men (10% of all cancer cases) and the second most common in women (9.4% of all cancer cases) making it the fourth highest cause of cancer death worldwide. Being a highly heterogeneous and aggressive disease, rate of its early detection is very less and incidence of rapid malignant proliferation is high. Further identification of new targets to restrict CRC progression is an absolute need of the hour. Interestingly, a novel molecule, NOSTRIN, shown to be anti-angiogenic, anti-invasive and anti-inflammatory, is abundantly expressed in colon and according to the Human Protein Atlas its expression is also detected in colorectal cancer samples both in RNA and protein levels. Furthermore, NOSTRIN has been demonstrated as a negative regulator of disease aggressiveness in pancreatic cancer patients and its increased expression was found to be associated with increased survival of patients. However, so far there has been no report directly linking NOSTRIN with colorectal cancer. We hypothesized that NOSTRIN plays a significant role in regulating a) colon cancer aggressiveness & b) cancer cell stemness. In our study, NOSTRIN expression was found to be inversely related to increased aggressiveness of colon cancer cell lines both in transcript and protein levels. Stable ectopic overexpression of NOSTRIN in CRC cell line led to decrease in expression of gene signatures for Epithelial to Mesenchymal Transition (EMT), one of the hallmark events of cancer, as established by our EMT array. This array data was further validated by western blotting. In line with this finding, migration and invasion ability of CRC cells were curbed by NOSTRIN overexpression as demonstrated by scratch wound and cell invasion assay, respectively. Furthermore, NOSTRIN overexpression remarkably decreased anchorage independent growth ability of CRC cells as shown by soft agar colony formation assay. These results indicate NOSTRIN reduces the metastatic potential of CRC cell lines. Interestingly, BrdU incorporation was significantly reduced in CRC cells by presence of excess NOSTRIN. In addition, heightened NOSTRIN expression led to decreased colonosphere formation and reduced expression of stemness markers, such as, CD133, CD44 and EpCAM by CRC cell lines. NOSTRIN's interaction with Cyclin Dependent Kinase1 (CDK1) and increased inhibitory CDK1 phosphorylation (Y15 and T14) in NOSTRIN over-expressing CRC cell lines might contribute to its inhibitory role in CRC cells’ stemness. Finally, a clear negative correlation between NOSTRIN expression and CRC disease progression was demonstrated in human patient samples using CRC cDNA array. Taken together, this report established a significant role of NOSTRIN in inhibiting a) EMT, b) metastatic potential and c) self-renewal of CRC cells.