Identifizierung und Charakterisierung spezifischer Liganden des medullären Schilddrüsenkarzinoms zur Entwicklung tumorselektiver Adenoviren

Die Gentherapie bietet bei der Tumorintervention eine alternative Behandlungsmethode zu herkömmlichen Therapien. Ziel der Gentherapie ist es, ein therapeutisches Gen in eine Zelle zu schleusen. Den effizientesten Gentransfer leisten virale Vektorsysteme, von denen neben Retroviren am häufigsten Adenoviren verwendet werden. Adenoviren sind in ihrer Zielgerichtetheit für das zu behandelnde Organ allerdings stark beschränkt, so dass es notwendig ist, ihren natürlichen Tropismus zu verbessern, damit sie selektiv das zu behandelnde Zielgewebe erreichen. Ein selektiver Tropismus kann in Form von spezifischen Liganden in das virale Kapsid integriert werden. Das in dieser Arbeit verwendete Zielgewebe ist das medulläre Schilddrüsenkarzinom (MTC). Mit Hilfe der sogenannten "Phage display"-Technik konnten für dieses Zielgewebe zwei spezifische Liganden in Form kurzer Peptidsequenzen, die auf der Phagenoberfläche expremiert werden, identifiziert werden. Für humane MTCs konnte die Sequenz HTFEPGV in vitro und in vivo, für murine MTC-Primärtumoren die Sequenz SRESPHP in vivo isoliert werden. Beide Sequenzen wiesen bei ihrer Charakterisierung eine hohe Affinität zum Zielgewebe, hohe Selektivität und die Fähigkeit zur Internalisierung auf. Zusätzlich konnte für die Sequenz HTFEPGV auch im adenoviralen Kontext die spezifische Bindung an die Zielzellen nachgewiesen werden. Ein weiterer Schwerpunkt dieser Arbeit war die genetische Modifizierung eines adenoviralen Vektors mittels zielzellspezifischer Peptide. Aus diesem Grund wurde eine Strategie zur Subklonierung der identifizierten kodierenden Peptidsequenzen entwickelt, auf deren Grundlage nun weitere in vivo Untersuchungen folgen können

Divergent mechanisms underlie Smad4-mediated positive regulation of the three genes encoding the basement membrane component laminin-332 (laminin-5)

<p>Abstract</p> <p>Background</p> <p>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 α3-, β3- and γ2-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 TGFβ superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFβ-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2.</p> <p>Methods</p> <p>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 TGFβ 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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Uncoupled responses of Smad4-deficient cancer cells to TNFα result in secretion of monomeric laminin-γ2

<p>Abstract</p> <p>Background</p> <p>Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-γ2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFα on tumor cells.</p> <p>Results</p> <p>Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFα with an increased expression of all three chains encoding laminin-332; coincubation with TGFβ and TNFα leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFα can induce expression of the γ2 and β3 but not the α3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of γ2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble γ2 is associated with increased cell migration.</p> <p>Conclusions</p> <p>Loss of Smad4 may lead to uncoupled induction of laminin-γ2 in response to TNFα and may therefore represent one of the mechanisms which underlie accumulation of laminin-γ2 at the invasive margin of a tumor. The finding, that γ2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.</p

Effects of transforming growth factor-β in the development of inflammatory pseudotumour-like lesions in a murine model

Alterations in transforming growth factor (TGF)-β signalling have been frequently implicated in human cancer, and an important mechanism underlying its pro-oncogenic nature is suppression of the host antitumour immune response. Considering the immunosuppressive effect of TGF-β, we asked whether human tumour cells, known to secrete TGF-β in culture, would survive and grow when implanted into the peritoneal cavity of immunocompetent mice. Therefore, we developed a xenogeneic model where mice were intraperitoneally (i.p.) injected with a TGF-β-secreting human colorectal adenocarcinoma cell line, LISP-A10. Although animals did not develop macroscopic tumours, the recovery and isolation of human tumour cells was achieved when an inflammatory environment was locally induced by the administration of complete Freund's adjuvant (CFA). This procedure significantly increased TGF-β concentrations in the peritoneal fluid and was accompanied by impaired activation of the host-specific immune response against LISP-A10 cells. Furthermore, inflammatory lesions resembling human inflammatory pseudotumours (IPTs) were observed on the surface of i.p. organs. These lesions could be induced by either injection of LISP-A10 cells, cells-conditioned medium or recombinant TGF-β but only after administration of CFA. In addition, host cyclooxygenase-2 and kinin receptors played an important role in the induction of TGF-β-mediated IPT-like lesions in our experimental model