Influence of the chemokine CXCL12 on the progression and the signaling in colorectal cancer

Das Chemokin CXCL12 (auch bekannt als SDF-1) ist ein kleines Protein (8-14) KDa, das in sechs Isoformen exprimiert wird (SDF-1α, SDF-1β, SDF-1γ, SDF- 1δ, SDF-1ε und SDF-1θ) von einem einzigen Gen, dass die Leukozyten-Wanderung regelt und variabel in einer Reihe von normalen und Krebsgeweben exprimiert wird.rnCXCL12 spielt verschiedene Rollen in der Tumorpathogenese. Es wurde nachgewiesen, dass CXCL12 das Tumorwachstum und die Malignität fördert, die Tumorangiogenese stärkt, sich an der Metastasierung beteiligt und zu immunsuppressiven Netzwerken innerhalb des Tumormikromilieus beiträgt. Daher liegt es nahe, dass der CXCL12/CXCR4-Signalweg ein wichtiges Ziel ist für die Entwicklung von neuartigen Krebstherapien.rnUm Licht auf die Rolle der Chemokin CXCL12 Splicevarianten in der Entwicklung von Krebs zu werfen und die mögliche physiologische Relevanz und ihre möglichen funktionellen Unterschiede bei Darmkrebs zu verstehen, haben wir alle CXCL12 Splicevarianten (alpha, beta, gamma, delta, epsilon und theta) in die kolorektalen Zelllinie SW480 und die Melanomzellinie D05 transfiziert und exprimiert.rnrnDiese Arbeit wurde erstellt, um die folgenden Ziele zu erreichen. Untersuchung der Rolle von CXCL12 Splicevarianten bei der Vermittlung von Tumorprogression, Adhäsion, Migration, Invasion und Metastasierung von Darmkrebs. Untersuchung, ob die CXCL12 Variantenwege ein wichtiges Ziel für die Entwicklung von Krebstherapien darstellen.rn• Um eine in vivo Mausmodell zu entwickeln, um die Rolle der CXCL12 Varianten im Rahmen des Tumorwachstums zu verstehen.rnrnUnsere Ergebnisse zeigen, dass:Der CXCL12 G801A Polymorphismus ist ein Low-Penetranz Risikofaktor für die Entwicklung von Darmkrebs. Der CXCL12-Gen-Polymorphismus rs1801157 ist mit dem T-Status (Tumor-node-Metastasen) assoziiert. Es gab keine Beziehung zwischen CXCL12-Gen-Polymorphismus rs1801157 und Fernmetastisen oder LN metastasen. Alle sechs CXCL12 Splicevarianten werden im Darmkrebs und in gesunder Kolon mucosa exprimiert. Die höchste Expression wird bei SDF-1alpha, dann SDF-1 beta gefunden. Alle sechs CXCL12 Varianten zeigen erhöhte Tumorzellproliferation in vitro. SDF-1beta, gefolgt von SDF-1alpha zeigte die größte Aktivität im Proliferationsassay.rn• Alle sechs CXCL12 Varianten induzieren die Tumorzelladhäsion.SDF-1beta dann SDF-1alpha zeigte die größte Aktivität im Rahmen des Adhäsionsassay. Alle sechs CXCL12 Varianten erhöhten die Zellmigration und Invasion von Tumorzellen in vitro. SDF-1theta und SDF-1epsilon 1theta zeigten die größte Aktivität, während die schwächste Aktivität mit SDF-1alpha und SDF-1beta beobachtet wurde. Alle sechs CXCL12 Varianten aktivieren Akt und (MAPK) Mitogen- acktivatedierte Protein kinase Wege und damit die Regulierung viele essentieller Prozesse in Tumorzellen, wie Proliferation, Migration, Invasion und Adhäsion. Es ist interessant festzustellen, dass AMD3100 die CXCL12 Splicevarianten inhibriert, die AKT-MEK-1/2-Phosphorylierung induzieren.rnDer Inhibitor AMD3100 unterdrückt stark die CXCL12 Varianten -delta, -epsilon und theta-und unterdrückt schwach CXCL12-gamma. während es keine signifikante Wirkung auf CXCL12-alpha und beta hatte. Es hat möglicherweise Auswirkungen auf mehrere große Signalwage in Bezug auf Proliferation, Migration und Invasions.rn• Es ist wichtig anzumerken, dass die Hemmung von CXCL12-Varianten durch AMD3100 einen der möglichen Ansaätze in der Krebstherapie darstellen kann.Wir schlagen vor, dass weitere Studien erwogen werden, die wir brauchen, um die biologische Aktivität dieser neuen CXCL12 Varianten bei verschiedenen Arten von Krebs klar zu verstehen.

Functional characterization of the placenta specific protein PLAC1 and its use for cancer immunotherapy

Summary Antibody-based cancer therapies have been successfully introduced into the clinic and have emerged as the most promising therapeutics in oncology. The limiting factor regarding the development of therapeutical antibody vaccines is the identification of tumor-associated antigens. PLAC1, the placenta-specific protein 1, was categorized for the first time by the group of Prof. Sahin as such a tumor-specific antigen. Within this work PLAC1 was characterized using a variety of biochemical methods. The protein expression profile, the cellular localization, the conformational state and especially the interacting partners of PLAC1 and its functionality in cancer were analyzed. Analysis of the protein expression profile of PLAC1 in normal human tissue confirms the published RT-PCR data. Except for placenta no PLAC1 expression was detectable in any other normal human tissue. Beyond, an increased PLAC1 expression was detected in several cancer cell lines derived of trophoblastic, breast and pancreatic lineage emphasizing its properties as tumor-specific antigen. rnThe cellular localization of PLAC1 revealed that PLAC1 contains a functional signal peptide which conducts the propeptide to the endoplasmic reticulum (ER) and results in the secretion of PLAC1 by the secretory pathway. Although PLAC1 did not exhibit a distinct transmembrane domain, no unbound protein was detectable in the cell culture supernatant of overexpressing cells. But by selective isolation of different cellular compartments PLAC1 was clearly enriched within the membrane fraction. Using size exclusion chromatography PLAC1 was characterized as a highly aggregating protein that forms a network of high molecular multimers, consisting of a mixture of non-covalent as well as covalent interactions. Those interactions were formed by PLAC1 with itself and probably other cellular components and proteins. Consequently, PLAC1 localize outside the cell, where it is associated to the membrane forming a stable extracellular coat-like structure.rnThe first mechanistic hint how PLAC1 promote cancer cell proliferation was achieved identifying the fibroblast growth factor FGF7 as a specific interacting partner of PLAC1. Moreover, it was clearly shown that PLAC1 as well as FGF7 bind to heparin, a glycosaminoglycan of the ECM that is also involved in FGF-signaling. The participation of PLAC1 within this pathway was approved after co-localizing PLAC1, FGF7 and the FGF7 specific receptor (FGFR2IIIb) and identifying the formation of a trimeric complex (PLAC1, FGF7 and the specific receptor FGFR2IIIb). Especially this trimeric complex revealed the role of PLAC1. Binding of PLAC1 together with FGF7 leads to the activation of the intracellular tyrosine kinase of the FGFR2IIIb-receptor and mediate the direct phosphorylation of the AKT-kinase. In the absence of PLAC1, no FGF7 mediated phosphorylation of AKT was observed. Consequently the function of PLAC1 was clarified: PLAC1 acts as a co-factor by stimulating proliferation by of the FGF7-FGFR2 signaling pathway.rnAll together, these novel biochemical findings underline that the placenta specific protein PLAC1 could be a new target for cancer immunotherapy, especially considering its potential applicability for antibody therapy in tumor patients.

Molecular mechanisms of shikonin and its derivatives in cancer therapy

The identification of molecular processes involved in cancer development and prognosis opened avenues for targeted therapies, which made treatment more tumor-specific and less toxic than conventional therapies. One important example is the epidermal growth factor receptor (EGFR) and EGFR-specific inhibitors (i.e. erlotinib). However, challenges such as drug resistance still remain in targeted therapies. Therefore, novel candidate compounds and new strategies are needed for improvement of therapy efficacy. Shikonin and its derivatives are cytotoxic constituents in traditional Chinese herbal medicine Zicao (Lithospermum erythrorhizin). In this study, we investigated the molecular mechanisms underlying the anti-cancer effects of shikonin and its derivatives in glioblastoma cells and leukemia cells. Most of shikonin derivatives showed strong cytotoxicity towards erlotinib-resistant glioblastoma cells, especially U87MG.ΔEGFR cells which overexpressed a deletion-activated EGFR (ΔEGFR). Moreover, shikonin and some derivatives worked synergistically with erlotinib in killing EGFR-overexpressing cells. Combination treatment with shikonin and erlotinib overcame the drug resistance of these cells to erlotinib. Western blotting analysis revealed that shikonin inhibited ΔEGFR phosphorylation and led to corresponding decreases in phosphorylation of EGFR downstream molecules. By means of Loewe additivity and Bliss independence drug interaction models, we found erlotinb and shikonin or its derivatives corporately suppressed ΔEGFR phosphorylation. We believed this to be a main mechanism responsible for their synergism in U87MG.ΔEGFR cells. In leukemia cells, which did not express EGFR, shikonin and its derivatives exhibited even greater cytotoxicity, suggesting the existence of other mechanisms. Microarray-based gene expression analysis uncovered the transcription factor c-MYC as the commonly deregulated molecule by shikonin and its derivatives. As validated by Western blotting analysis, DNA-binding assays and molecular docking, shikonin and its derivatives bound and inhibited c-MYC. Furthermore, the deregulation of ERK, JNK MAPK and AKT activity was closely associated with the reduction of c-MYC, indicating the involvement of these signaling molecules in shikonin-triggered c-MYC inactivation. In conclusion, the inhibition of EGFR signaling, synergism with erlotinib and targeting of c-MYC illustrate the multi-targeted feature of natural naphthoquinones such as shikonin and derivatives. This may open attractive possibilities for their use in a molecular targeted cancer therapy.