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  • 1
    Keywords: EXPRESSION ; PROTEINS ; COLORECTAL-CANCER ; EPITHELIAL-CELLS ; PROTEOMICS ; TUMOR-DERIVED EXOSOMES ; DIAGNOSTIC BIOMARKERS ; MICROVESICLES ; COMMUNICATION ; CD73
    Abstract: BACKGROUND: Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. RESULTS: Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. CONCLUSION: The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.
    Type of Publication: Journal article published
    PubMed ID: 25469109
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  • 2
    Keywords: CANCER ; CELLS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; carcinoma ; Germany ; INHIBITION ; GENE ; PROTEIN ; PROTEINS ; LINES ; INFECTION ; MECHANISM ; prognosis ; CARCINOGENESIS ; mechanisms ; CELL-LINES ; E7 ; DELETION ; LESIONS ; PROGRESSION ; CARCINOMA CELLS ; WOMEN ; COLORECTAL-CANCER ; CERVICAL-CANCER ; LINE ; TUMOR-SUPPRESSOR GENE ; human papillomavirus ; CARCINOMA-CELLS ; BETA ; CERVICAL-CARCINOMA ; CARCINOMAS ; squamous cell carcinoma ; intraepithelial neoplasia ; UTERINE CERVIX ; GROWTH-FACTOR-BETA ; POOR-PROGNOSIS ; cell lines ; GENOMIC INSTABILITY ; FACTOR-BETA ; molecular ; DEFICIENCY ; TUMOR-SUPPRESSOR ; TUMOR-GROWTH ; TGF-BETA ; MOLECULAR-MECHANISMS ; RESPONSIVENESS ; carcinoma cell ; CIN lesion ; cytogenetic ; DPC4 INACTIVATION ; multistep carcinogenesis ; PAPILLOMAVIRUS INFECTIONS ; SUPPRESSOR ; TGF beta ; tumor suppressor gene
    Abstract: Squamous cell carcinoma of the uterine cervix is one of the most frequent cancers affecting women worldwide. Carcinomas arise from cervical intraepithelial lesions, in which infection with high-risk human papillomavirus types has led to deregulated growth control through the actions of the viral E6 and E7 oncoproteins. The molecular mechanisms underlying progression to invasive tumor growth are poorly understood. One important feature, however, is the escape from growth inhibition by transforming growth factor beta (TGF-beta). Loss of chromosomal arm 18q is among the most frequent cytogenetic alterations in cervical cancers and has been associated with poor prognosis. Since the TGF-beta response is mediated by Smad proteins and the tumor suppressor gene Smad4 resides at 18q21, we have analysed the Smad4 gene for cervical cancer-associated alterations in cell lines and primary carcinomas. Here, we report Smad4 deficiency in four out of 13 cervical cancer cell lines which is due to an intronic rearrangement or deletions of 30 exons. All cell lines, however, showed either absent or moderate responsiveness to TGF-beta irrespective of their Smad4 status. In 41 primary squamous cervical carcinomas analysed, 10 samples showed loss of Smad4 protein expression and 26 samples a reduced expression. Altogether, our results strongly suggest that Smad4 gene alterations are involved in cervical carcinogenesis
    Type of Publication: Journal article published
    PubMed ID: 15531914
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  • 3
    Keywords: CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; GROWTH ; INVASION ; tumor ; TUMOR-CELLS ; CELL ; Germany ; human ; SITE ; SITES ; GENE ; GENES ; transcription ; COMPONENTS ; MOLECULES ; TISSUE ; MECHANISM ; FAMILY ; TRANSCRIPTION FACTOR ; IMPACT ; CARCINOGENESIS ; INDUCTION ; mechanisms ; BINDING ; SIGNAL ; MOLECULE ; ALPHA ; cytokines ; TARGET ; DELETION ; CHROMATIN ; PROMOTER ; MEMBRANE ; PROMOTERS ; MUTATION ; inactivation ; DERIVATIVES ; REGION ; CANCER-CELLS ; REGIONS ; MUTATIONS ; BETA ; SUPERFAMILY ; GROWTH-FACTOR-BETA ; TRANSCRIPTIONAL REGULATION ; GAMMA-2 CHAIN ; CYTOKINE ; molecular ; ONCOLOGY ; FAMILIES ; TUMOR SUPPRESSION ; TUMOR-SUPPRESSOR ; basement membrane ; TRANSFECTION ; TGF-BETA ; interaction ; MOLECULAR-MECHANISMS ; methods ; SUPPRESSOR ; TGF beta ; SIGNALS ; COLON-CARCINOMA CELLS ; BARRIER ; ENGLAND ; UPSTREAM ; response ; synthesis ; Smad4 ; SUPPRESSOR E-CADHERIN ; chromatin immunoprecipitation ; tumor suppressor ; FUNCTIONAL INACTIVATION ; BINDING SITE ; ACTIVATOR PROTEIN-1 ; AP-1 COMPLEX
    Abstract: Background: Functional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane ( BM) barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5), a heterotrimeric BM component composed of alpha 3-, beta 3- and gamma 2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGF beta superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGF beta-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2. Methods: Promoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGF beta induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation. Results: Herein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1) sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters. Conclusion: We hypothesize that this divergence in modular regulation of the three promoters may lay the ground for uncoupled regulation of Laminin-332 in Smad4-deficient tumor cells in response to stromally expressed cytokines acting on budding tumor cells
    Type of Publication: Journal article published
    PubMed ID: 18664273
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  • 4
    Keywords: CELLS ; EXPRESSION ; carcinoma ; FACTOR RECEPTOR ; IN-VIVO ; PROTEIN ; HEAD ; NECK ; pancreatic cancer ; PROTEOMICS ; EPIDERMAL-GROWTH-FACTOR ; SERUM ; biomarker ; exosome ; METASTATIC BREAST-CANCER ; TARGETED THERAPY ; EGFR forms ; Secretome ; TUMOR AGGRESSIVENESS
    Abstract: Aims: Members of the epidermal growth factor receptor (EGFR) family represent validated targets for anticancer therapy and EGFR inhibitors have also shown efficacy in pancreatic carcinoma. We here described in detail molecular forms of the EGF receptor released by pancreatic cancer cells. We found peptides specific for the EGER in the secretomes of five pancreatic cancer cell lines. Secretomes from cultured cancer cells are widely used as sources for serum biomarker discovery. Main methods: The detailed analysis of EGFR forms in secretomes of human pancreatic cancer cells is a compilation of results from mass spectrometry (MS) and Western blotting with intracellular and extracellular domain specific antibodies. Key findings: Pancreatic cancer cells secrete a 110 kDa soluble form of the EGFR (sEGFR) representing the ligand binding extracellular EGFR domains and presumably released by ectodomain shedding. At the same time, as constituents of exosomes, the EGFR is released as full-length intact receptor (170 kDa) and as a 65 kDa processed form, the C-terminal remnant fragment that corresponds to the intracellular kinase domain. Significance: The detailed characterization of diverse EGER forms released by pancreatic cancer cells in vitro and presumably in vivo bears important implications for functional studies, for the validation of soluble EGFR as a serum biomarker and for the design of targeted therapies.
    Type of Publication: Journal article published
    PubMed ID: 21763319
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  • 5
  • 6
    Keywords: CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; tumor ; carcinoma ; CELL ; Germany ; human ; IN-VIVO ; MODEL ; VITRO ; VIVO ; SYSTEM ; GENE ; GENE-EXPRESSION ; GENES ; cell line ; TRANSDUCTION ; RESPONSES ; IMPACT ; INDUCTION ; tumour ; DOWN-REGULATION ; SUPPRESSION ; SIGNAL ; cytokines ; TARGET ; gene expression ; resistance ; CARCINOMA CELLS ; colorectal cancer ; COLORECTAL-CANCER ; cervical cancer ; CERVICAL-CANCER ; CELL-LINE ; LINE ; SIGNALING PATHWAY ; CANCER-CELLS ; COLORECTAL CANCERS ; EXTRACELLULAR-MATRIX ; CARCINOMA-CELLS ; SUPERFAMILY ; CERVICAL-CARCINOMA ; cervical carcinoma ; GROWTH-FACTOR-BETA ; TARGETS ; C-MYC ; OVEREXPRESSION ; pancreatic carcinoma ; HIGH-LEVEL ; CELL-GROWTH ; CYTOKINE ; MATRIX ; ONCOLOGY ; TUMOR SUPPRESSION ; LIGHT ; extracellular matrix ; RESTORATION ; regulation ; TGF-BETA ; interaction ; LEVEL ; TARGET GENES ; methods ; pancreatic ; SUPPRESSOR ; EVENTS ; tumour suppressor ; function ; LOSSES ; CANCERS ; in vivo ; SIGNALS ; carcinogenic ; COLON-CARCINOMA CELLS ; ENGLAND ; PREDICT ; colorectal ; NOV ; evidence ; cell growth ; BETAIG-H3 GENE ; BRONCHIAL EPITHELIAL-CELLS ; Smad4 ; STABLE RNA INTERFERENCE ; SUPPRESSOR E-CADHERIN ; tumour suppression
    Abstract: Background: Smad4 is a tumour suppressor frequently inactivated in pancreatic and colorectal cancers. We have recently reported loss of Smad4 in every fourth carcinoma of the uterine cervix. Smad4 transmits signals from the TGF-beta superfamily of cytokines and functions as a versatile transcriptional co-modulator. The prevailing view suggests that the tumour suppressor function of Smad4 primarily resides in its capability to mediate TGF-beta growth inhibitory responses. However, accumulating evidence indicates, that the acquisition of TGF-beta resistance and loss of Smad4 may be independent events in the carcinogenic process. Through inducible reexpression of Smad4 in cervical cancer cells we wished to shed more light on this issue and to identify target genes implicated in Smad4 dependent tumor suppression. Methods: Smad4-deficient human C4-II cervical carcinoma cells were used to establish inducible Smad4 reexpression using the commercial Tet-on (TM) system (Clontech). The impact of Smad4 reexpression on cell growth was analysed in vitro and in vivo. Transcriptional responses were assessed through profiling on cDNA macroarrays (Clontech) and validated through Northern blotting. Results: Clones were obtained that express Smad4 at widely varying levels from approximately physiological to 50-fold overexpression. Smad4-mediated tumour suppression in vivo was apparent at physiological expression levels as well as in Smad4 overexpressing clones. Smad4 reexpression in a dose-dependent manner was associated with transcriptional induction of the extracellular matrix-associated genes, BigH3, fibronectin and PAI-I, in response to TGF-beta. Smad4-dependent regulation of these secreted Smad4 targets is not restricted to cervical carcinoma cells and was confirmed in pancreatic carcinoma cells reexpressing Smad4 after retroviral transduction and in a stable Smad4 knockdown model. On the other hand, the classical cell cycle-associated TGF-beta target genes, c-myc, p21 and p15, remained unaltered. Conclusion: Our results show that Smad4-mediated tumour suppression in cervical cancer cells is not due to restoration of TGF-beta growth inhibitory responses. Rather, tumour cell-ECM interactions may be more relevant for Smad4-mediated tumour suppression. C4-II cells with a high level inducible Smad4 expression may serve as a model to indicate further Smad4 targets responsive to diverse environmental stimuli operative in vivo
    Type of Publication: Journal article published
    PubMed ID: 17997817
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  • 7
    Keywords: CELLS ; EXPRESSION ; GROWTH ; INVASION ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; human ; COMMON ; GENE ; GENE-EXPRESSION ; GENES ; TUMORS ; TRANSDUCTION ; MECHANISM ; CARCINOGENESIS ; colon ; mechanisms ; BIOLOGY ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; MOLECULE ; cytokines ; TARGET ; STAGE ; PROGRESSION ; MALIGNANCIES ; ENCODES ; MEMBRANE ; METASTASIS ; genetics ; COLORECTAL-CANCER ; COMPONENT ; inactivation ; EXTRACELLULAR-MATRIX ; ONCOGENE ; SUPERFAMILY ; CARCINOMAS ; beta-catenin ; GROWTH-FACTOR-BETA ; TARGETS ; TUMOR CELLS ; heredity ; REGULATOR ; FACTOR-BETA ; GAMMA-2 CHAIN ; CYTOKINE ; molecular biology ; molecular ; MATRIX ; CHAIN ; MALIGNANCY ; ONCOLOGY ; pancreas ; TUMOR-SUPPRESSOR ; basement membrane ; BASEMENT-MEMBRANE ; extracellular matrix ; secretion ; LEADS ; TRANSITION ; TGF-BETA ; pancreatic ; TUMOR-CELL ; SUPPRESSOR ; function ; COLON-CARCINOMA CELLS ; ENGLAND ; RECONSTITUTION ; LAMININ-5 ; CASCADE ; OCCURS ; Smad4 ; STABLE RNA INTERFERENCE ; DPC4 ; tumor suppressor Smad4
    Abstract: The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smadsignal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane ( BM), a specialized sheet of extracellular matrix produced through of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data de. ne the expression control of an essential BM component as a novel function for the tumor suppressor Smad4
    Type of Publication: Journal article published
    PubMed ID: 16953227
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  • 8
    Keywords: EXPRESSION ; PROTEINS ; BIOMARKERS ; Drosophila ; TUMOR-SUPPRESSOR GENE ; SIGNALING PATHWAY ; beta-catenin ; ADHESION MOLECULE ; SERUM ; PROTOCADHERIN FAT1
    Abstract: In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.
    Type of Publication: Journal article published
    PubMed ID: 24625754
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