Abstract
The proprotein convertases (PCs) are responsible for the maturation of precursor proteins, and are involved in multiple and critical biological processes. Over the past 30 years, the PCs have had great translational applications, but the physiological roles of PC7, the seventh member of the family, are still obscure. Searching for new substrates of PC7, a quantitative proteomics screen for selective enrichment of N-glycosylated polypeptides secreted from hepatic HuH7 cells identified two human type-II transmembrane proteins of unknown function(s): Cancer Susceptibility Candidate 4 (CASC4) and Golgi Phosphoprotein of 130 kDa (GPP130/GOLIM4). Concentrating on CASC4, its mutagenesis characterized the PC7/Furin-shedding site to occur at KR66↓NS, in HEK293 cells. We defined PC7 and Furin trafficking and activity, and demonstrated that CASC4 shedding occurs in acidic endosomes and/or in the trans-Golgi Network. Our data unraveled a cancer-protective role for CASC4, because siRNA silencing of endogenous CASC4 expression in the invasive triple-negative breast cancer human cell line MDA-MB-231 resulted in a significantly increased cellular migration and invasion. Conversely, MDA-MB-231 cells stably expressing CASC4 exhibited reduced migration and invasion, which can be explained by an increased number of paxillin-positive focal adhesions. This phenotypic cancer-protective role of CASC4 is reversed in cells overexpressing an optimally PC7/Furin-cleaved CASC4 mutant, or upon overexpression of the N-terminally convertase-generated membrane-bound segment. This phenotype was associated with increased formation of podosome-like structures, especially evident in cells overexpressing the N-terminal fragment. In accord, breast cancer patients’ data sets show that high CASC4 and PCSK7 expression levels predict a significantly worse prognosis compared to high CASC4 but low PCSK7 levels. In conclusion, CASC4 shedding not only disrupts its anti-migratory/invasive role, but also generates a membrane-bound fragment that drastically modifies the actin cytoskeleton, resulting in an enhanced cellular migration and invasion. This phenotype might be clinically relevant in the prognosis of breast cancer patients.
Highlights
The proprotein convertases (PCs) constitute a family of nine serine secretory proteases that regulate diverse biological processes in both health and disease states[1]
The PCs have key roles in both health and disease states by cleavage of precursor proteins[1], which results in the bioactivation of proteins, but sometimes may generate cleaved products endowed with novel functions[44,45]
One of the specific functions of PC7 is the shedding of the human type-II TfR19, resulting in the secretion of a circulating sTfR1 that correlates with iron deficiency[50]
Summary
The proprotein convertases (PCs) constitute a family of nine serine secretory proteases that regulate diverse biological processes in both health and disease states[1]. Duval et al Cell Death and Disease (2020)11:665 proteins at specific single or paired basic amino acid (aa) within the motif (R/K)-(2Xn)-(R/K)↓, where Xn = 0–3 spacer aa[2] Because of their roles in the processing of many critical secretory substrates, e.g., activation of TGFβ3 and matrix metalloproteases[4], PCs, such as Furin, PC5, PACE4, and PC7 were implicated in cancer/metastasis[5,6,7]. To better understand the PC7 biology and pathophysiology, we undertook an unbiased quantitative proteomics screen of N-glycosylated secreted products from hepatic HuH7 cells overexpressing PC7 This screen led us to identify two shed type-II transmembrane proteins of unknown biological functions: Cancer Susceptibility Candidate 4 (CASC4)[13], and Golgi Phosphoprotein of 130 kDa (GPP130/GOLIM4)[14]. A significant increase in secreted (shed) sCASC4 was found upon analysis of the N-glycosylated secretome from highly metastatic breast cancer cell lines[17]
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