We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time-and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2-mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2-mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.

CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKC␦ to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein. CUB-domain-containing protein 1 (CDCP1)3 is an 836 amino acid integral membrane glycoprotein with a type I orientation at the cell surface (1-4), that is up-regulated in a number of malignancies including breast (1, 5, 6), colon (1, 2, 7), and lung (1) cancers. Of potential clinical significance, CDCP1 expression correlates with recurrence and patient survival rate in renal cell carcinomas (8) and lung adenocarcinomas (9), indicating that it may be suitable as a prognostic marker. Consistent with a role in cancer progression, silencing of CDCP1 reduced the metastatic ability of lung cancer A549 cells (10) and the peritoneal dissemination of gastric cancer 44As3 cells (11) in mice. Although its biological function is not known, the potential of CDCP1 as a therapeutic target for cancer treatment has been highlighted by studies showing that antibody-mediated inhibition of CDCP1 reduced metastasis of prostate cancer PC3 cells in mice (12, 13) and chicken embryos (13). Currently the mechanisms regulating CDCP1 in cancer and normal physiology are not well defined (14).During cellular processing, the 29-residue CDCP1 N-terminal signal peptide is removed generating a protein with molecular mass identified as either 135 kDa (2, 10, 15) or 140 kDa (3, 4) that contains 30 -40 kDa of N-linked glycans (2). In addition to this full-length form, there is evidence that a shorter CDCP1 species is expressed endogenously by a range of cell lines or is generated through the action of exogenous serine proteases. For example, lung cancer A549, PC14, H520, H322, and H157 cells (10) and gastric cancer 44As3 and 58As9 cells (11) resuspended non-enzymatically ...

Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product-specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.

SummaryIn the last few years dysregulated expression of the cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) has been associated with several cancers and this cell surface molecule has been recognized both as a tumor marker and as a potential target to disrupt progression of cancer. Here we summarize what is known about CDCP1 including its structural features, expression in normal and cancerous tissues, and the in vitro experiments and studies in animal models that have provided the key insights into its potential role in tumor formation and metastasis in humans. We conclude by highlighting opportunities and challenges in targeting CDCP1 in cancer.2009 IUBMB IUBMB Life, 61(7): [723][724][725][726][727][728][729][730] 2009

The fungus Emericella nidulans var. acristata was isolated as an endophyte from a Mediterranean green alga. Cultivation of this fungus yielded two new compounds, arugosins G (1) and H (2), together with the known metabolites 3-9. Arugosins (1-4) are benzophenone derivatives, biosynthetically related to the xanthones 5, 6, and 9. The indole alkaloid 7 displayed antitumor activity in a panel of 36 human tumor cell lines, exhibiting a mean IC(50) value of 5.5 microg/mL in an in vitro survival and proliferation assay. Furthermore, compounds 3 and 4 showed moderate antitumor activity toward individual tumor cell lines. None of compounds 1-8 exhibited any immunostimulatory activity assessed as the capacity to induce cytokines in PBMCs from healthy donors.

This paper introduces the new robot programming language LightRocks(Light Weight Robot Coding for Skills), a domain specific language (DSL) for robot programming. The language offers three different level of abstraction for robot programming. On lowest level skills are coded by domain experts. On a more abstract level these skills are supposed to be combined by shop floor workers or technicians to define tasks. The language is designed to allow as much flexibility as necessary on the lowest level of abstraction and is kept as simple as possible with the more abstract layers. A Statechart like model is used to describe the different levels of detail. For this we apply the UML/P and the language workbench MontiCore. To this end we are able to generate code while hiding controller specific implementation details. In addition the development in LightRocks is supported by a generic graphical editor implemented as an Eclipse plugin.

Industry 4.0 integrates Cyber-Physical Systems with the Internet of Things to optimize the complete value-added chain. Successfully applying Industry 4.0 requires the cooperation of various stakeholders from different domains. Domain-specific modeling languages promise to facilitate their involvement through leveraging (domain-specific) models to primary development artifacts. Objective: We aim to assess the use of modeling in Industry 4.0 through the lens of modeling languages in a broad sense. Method: Based on an extensive literature review, we updated our systematic mapping study on modeling languages and modeling techniques used in Industry 4.0 [159] to include publications until February 2018. Overall, the updated study considers 3344 candidate publications that were systematically investigated until 408 relevant publications were identified. Based on these, we developed an updated map of the research landscape on modeling languages and techniques for Industry 4.0. Results: Research on modeling languages in Industry 4.0 focuses on contributing methods to solve the challenges of digital representation and integration. To this end, languages from systems engineering and knowledge representation are applied most often but rarely combined. There also is a gap between the communities researching and applying modeling languages for In