TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2's cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

Introduction: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) benefit advanced lung adenocarcinoma (ADC) patients harboring activating EGFR mutations. We aimed to identify biomarkers to monitor and predict the progression of patients receiving EGFR-TKIs via a comprehensive omic analysis. Methods:We applied quantitative proteomics to generate the TKI resistance-associated pleural effusion (PE) proteome from ADC patients with or without EGFR-TKI resistance. Candidates were selected from integrated genomic and proteomic datasets. The PE (n=33) and serum (n=329) levels of potential biomarkers were validated with enzyme-linked immunosorbent assays (ELISAs). Western blotting was applied to detect protein expression in tissues, PEs and a cell line. Gene knockdown, TKI treatment and proliferation assays were used to determine EGFR-TKI sensitivity. Progression-free survival (PFS) and overall survival (OS) were assessed to evaluate the prognostic values of the potential biomarkers.Results: Fifteen proteins were identified as potential biomarkers of EGFR-TKI resistance. Cadherin-3 (CDH3) was overexpressed in ADC tissues compared to Research.

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ADP‐ribosylation factors (Arfs) and Arf‐like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP‐bound and inactive GDP‐bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase‐activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans‐Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP‐bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity‐deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull‐down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1‐GTP in cells expressing ArfGAP1‐myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome‐to‐TGN retrograde transport of the Shiga toxin B‐subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.

Lung adenocarcinoma (ADC) is the most common histological subtype of lung cancer, the leading cause of cancer‐related deaths worldwide. Epidermal growth factor receptor (EGFR) mutations strikingly target lung ADC in East Asia. EGFR tyrosine kinase inhibitor (EGFR‐TKI) has been developed as a targeted therapy for treatment of advanced lung ADC with activating EGFR mutations. Unfortunately, neither druggable targets nor useful biomarkers can be used to benefit ADC patients with wild‐type (WT) EGFR. We herein aimed to identify the differentially expressed phosphoproteins and kinases in ADC tissues with EGFR‐WT. We applied super‐SILAC combined with TiO2 phosphopeptide enrichment and liquid chromatography‐tandem mass spectrometry to establish the stage‐related phosphoproteome with 20244 phosphopeptides in ADC tissues. The bioinformatics analyses revealed the stage‐dependent phosphorylation profile, upstream signaling and the kinase cascade in ADC tissues. We also observed that the impact of EGF, PTK2B, JAK2, ICAM1, or VEGF on pathogenesis of the early‐ and late‐stage cancers was indeed different. To explore the clinical applications of these dysregulated phosphoproteins in lung ADC, the potential markers were selected for further verifications/validations via western blotting and immunohistochemistry staining.Support or Funding InformationMinistry of Science and Technology, Taiwan, R.O.C. (105‐2320‐B‐182‐035‐MY3) and Chang Gung Medical Research Fund (CMRPD1H0081, CMRPD1H0082 and BMRP894).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.