Ectopic expression of DOCK8 regulates lysosome-mediated pancreatic tumor cell invasion.

Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor growth and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 8 (DOCK8) on lysosomes. Surprisingly, DOCK8 is aberrantly expressed in a subset of PDAC, where it promotes cell invasion in vitro and in vivo. DOCK8 associates with lysosomes and regulates lysosomal morphology and motility, with loss of DOCK8 leading to increased lysosome size. DOCK8 promotes actin polymerization at the surface of lysosomes while also increasing the proteolytic activity of the lysosomal protease cathepsin B. Critically, depletion of DOCK8 significantly reduces cathepsin-dependent extracellular matrix degradation and impairs the invasive capacity of PDAC cells. These findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation and invasion in pancreatic cancer cells.

A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

A size-exclusion-based approach for purifying extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are released into blood from multiple organs and carry molecular cargo that facilitates inter-organ communication and an integrated response to physiological and pathological stimuli. Interrogation of the protein cargo of EVs is currently limited by the absence of optimal and reproducible approaches for purifying plasma EVs that are suitable for downstream proteomic analyses. We describe a size-exclusion chromatography (SEC)-based method to purify EVs from platelet-poor plasma (PPP) for proteomics profiling via high-resolution mass spectrometry (SEC-MS). The SEC-MS method identifies more proteins with higher precision than several conventional EV isolation approaches. We apply the SEC-MS method to identify the unique proteomic signatures of EVs released from platelets, adipocytes, muscle cells, and hepatocytes, with the goal of identifying tissue-specific EV markers. Furthermore, we apply the SEC-MS approach to evaluate the effects of a single bout of exercise on EV proteomic cargo in human plasma.

TBK1 regulates regeneration of pancreatic ß-cells.

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate ß-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in ß-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat ß-cells. Conversely, TBK1 overexpression decreased sensitivity of ß-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of ß-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of ß-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived ß-cells and human islets. TBK1 expression was increased in ß-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and ß-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional ß-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a ß-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional ß-cells.