Joshi, Neha

Gastric cancer is a leading cause of death from cancer globally. Gastric cancer is classified into intestinal, diffuse and indeterminate subtypes based on histology according to the Laurén classification. The intestinal and diffuse subtypes, although different in histology, demographics and outcomes, are still treated in the same fashion. This study was designed to discover proteomic signatures of diffuse and intestinal subtypes. Mass spectrometry-based proteomics using tandem mass tags (TMT)-based multiplexed analysis was used to identify proteins in tumor tissues from patients with diffuse or intestinal gastric cancer with adjacent normal tissue control. A total of 7448 or 4846 proteins were identified from intestinal or diffuse subtype, respectively. This quantitative mass spectrometric analysis defined a proteomic signature of differential expression across the two subtypes, which included gremlin1 (GREM1), bcl-2-associated athanogene 2 (BAG2), olfactomedin 4 (OLFM4), thyroid hormone receptor interacting protein 6 (TRIP6) and melanoma-associated antigen 9 (MAGE-A9) proteins. Although GREM1, BAG2, OLFM4, TRIP6 and MAGE-A9 have all been previously implicated in tumor progression and metastasis, they have not been linked to intestinal or diffuse subtypes of gastric cancer. Using immunohistochemical labelling of a tissue microarray comprising of 124 cases of gastric cancer, we validated the proteomic signature obtained by mass spectrometry in the discovery cohort. Our findings should help investigate the pathogenesis of these gastric cancer subtypes and potentially lead to strategies for early diagnosis and treatment.

DKK3 is a secreted protein, which belongs to a family of Wnt antagonists and acts as a potential tumor suppressor in gallbladder cancer. To further understand its tumor suppressor functions, we overexpressed DKK3 in 3 GBC cell lines. We have employed high-resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides to check the downstream regulators. In this study, we reported for the first time the alteration in the phosphorylation of 14 kinases upon DKK3 overexpression. In addition, we observed DKK3 induced hyper phosphorylation of 2 phosphatases: PPP1R12A and PTPRA, which have not been reported previously. Canonical pathway analysis of altered molecules indicated differential enrichment of signaling cascades upon DKK3 overexpression in all the 3 cell lines. Protein kinase A signaling, Sirtuin signaling pathway, and Cell Cycle Control of Chromosomal Replication were observed to be differentially activated in the GBC cell lines. Our study revealed, DKK3 overexpression has differential effect based on the aggressive behavior of the cell lines. This study expands the understanding of DKK3-mediated signaling events and can be used as a primary factor for understanding the complex nature of this molecule.