Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MYC sensitises cells to apoptosis by driving energetic demand.

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

Identification of differentially regulated deceitful proteins in SH-SY5Y cells engineered with Tet-regulated protein expression system.

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.