Abstract
Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker β-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.
Highlights
Ubiquitin (Ub) is a highly conserved 76 amino-acid protein that is covalently conjugated to target proteins by the consecutive actions of three enzymes (E1, E2, E3) in a process known as ubiquitylation [1].Ub conjugation is an incredibly complex post-translational modification involved in the regulation of many cellular processes such as proteasome-mediated proteolysis, but it has various non-degradative functions, ranging from signal transduction to transcription, from endocytosis to protein trafficking, from DNA repair to cell survival and proliferation [1,2]
Our results demonstrate the role of UBB and UBC as pro-survival genes in primary Gastric cancer (GC) cell line 23132/87 and show that the combined silencing of these two Ub genes in the primary gastric adenocarcinoma cells led to a decrease in their viability, exerted through activation of the extrinsic pathway of apoptosis, and a reduction in levels of the oncoprotein β-catenin, which has a role in overproliferation, migration, invasion of various tumors and in the epithelial to mesenchymal transition (EMT) process [30]
Since LUCAT1 is highly expressed in gastric cancer [42], it can be hypothesized that a similar post-translational control occurs in 23132/87 and MKN45 gastric cancer cells
Summary
Ubiquitin (Ub) is a highly conserved 76 amino-acid protein that is covalently conjugated to target proteins by the consecutive actions of three enzymes (E1, E2, E3) in a process known as ubiquitylation [1].Ub conjugation is an incredibly complex post-translational modification involved in the regulation of many cellular processes such as proteasome-mediated proteolysis, but it has various non-degradative functions, ranging from signal transduction to transcription, from endocytosis to protein trafficking, from DNA repair to cell survival and proliferation [1,2]. UBB and UBC encode Ub linear polyproteins formed by three and nine Ub monomers, respectively [5], while UBA52 and RPS27A produce a fusion product where the C-terminus of one Ub molecule is fused to a ribosomal protein [6,7]. These precursors are co- and post-translationally processed in their mature forms by deubiquitinases (DUBs), which selectively cleave Ub monomers from their fusion partners [8]
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