Myoepithelial cell‐specific expression of stefin A as a suppressor of early breast cancer invasion

Abstract

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles

“…Raw data were pre‐processed as previously described and processed using MaxQuant(v1.6.0.16) with Andromeda (v1.5.6), using a Helicobacter pylori (UniProt, UP000000429, Jan 2018) reference sequence database. Data were searched as described with a parent tolerance of 10 ppm, fragment tolerance of 0.5 Da, and minimum peptide length 5, with false discovery rate 1% at the peptide and protein levels, tryptic digestion with up to two missed cleavages, cysteine carbamidomethylation as fixed modification, and methionine oxidation and protein N ‐terminal acetylation as variable modifications and data analyzed with label‐free quantitation (LFQ) . LFQ intensity values were normalized for protein length and fold change ratios calculated.…”

Proteomic and Post‐Translational Modification Profiling of Exosome‐Mimetic Nanovesicles Compared to Exosomes

“…M‐NVs, exosomes, and cell lysates were prepared for proteome profiling as described . Briefly, M‐NVs (20 µg) and exosomes (20 µg), were lysed in 100 m m triethylammonium bicarbonate in 0.1% Rapigest (Waters, Milford, MA) with 2 min sonication and incubation at 95 °C for 5 min.…”

“…Technical replicates were averaged and hierarchical clustering analyses (HCA) performed, missing values computations performed as described. For differential expression analyses between groups, unpaired two‐tailed t ‐tests and resulting p ‐values were adjusted by the Benjamini–Hochberg multitest adjustment method for a high number of comparisons and statistics performed as previously described . For gene ontology (GO) enrichment and pathway analyses, FunRich; v3.1.3, Kyoto Encyclopedia of Genes and Genomes (KEGG) and NIH Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resources 6.7 (DAVID) resources were utilized using recommended analytical parameters .…”

Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114

“…A total of two individual gel bands (≈5–6 mm each) were excised, representing the entire gel. Gel pieces were destained (50 m m ammonium bicarbonate/acetonitrile), reduced (10 m m DTT (Calbiochem) for 30 min), alkylated (50 m m iodoacetic acid (Fluka) for 30 min), and trypsinized (0.2 µg trypsin (Promega Sequencing Grade) for 16 h at 37 °C) . Generated tryptic peptides were extracted from the gel and purified using reversed‐phase C18 StageTips (Sep‐Pack cartridge, Waters, MA, USA) using 85% v/v acetonitrile (ACN) in 0.5% v/v formic acid (FA).…”