4.3.4 Validation of the array: Fgf10, FgfR2b, Etv4/5, Shh connection. Figure 18 displays the genes found in the groups Early 4, Late 4 as well as Late 1 and Late 3. The group Early 4 contains genes such as Sftpc and Id2 which are related to epithelial cell differentiation. It contains also Etv5 which has been shown to be positively regulated by FGF signaling. In the Late 1 group, Fgf10 is up-regulated between 6 and 9 hours after dox-IP, which support the decrease in SHH signaling. In the Late 3 group, the presence of Ptch2, Foxl1, Foxf1 and Gli1, suggesting decreased SHH signaling to the mesenchyme upon FGFR2b ligand inhibition between 9 hours and 6 hours after dox-IP. And finally, the group Late 4 contains genes that are linked to the epithelial differentiation such as Sftpa1, Sftpb, Sftpc as well as Hopx, it contains also sonic hedgehog (Shh) suggesting that Fgf10 controls Shh expression positively. Through our analysis, we can deduce that our data supports the model published by Herriges et al. where they reported that Fgf10, acting via FgfR2b in the epithelium, promotes the expression of the transcription factors Etv4 and Etv5, which in turn promote Shh expression. Shh is then secreted by the epithelium and binds to Ptch2 to release the inhibition of Smoothened (Smo). Smo in turn activates Gli1, and this last one activates the expression of Foxf1 and Foxl. Then, Shh signaling represses Fgf10 expression (Herriges et al., 2015). Our data revealed that by the attenuation of FGF10 signaling, Etv4/5, Shh, Ptch2, Gli1, Foxf1 and Foxl1 are all reduced, while Fgf10 is increased (Figure 18E). We propose that Fgf10 acts via FgfR2b, positively regulating the expression of Etv4/Etv5, which in turn regulates Shh expression, and therefore the downstream genes involved in the SHH pathway, validating the well-established FGF10-SHH regulatory feedback loop during lung
mammary tumors significantly faster than mice expressing either transgene alone. Histological analysis showed that ErbB2 tumors were predominantly adenocarcinomas, CD8- IGF-IR tumors were mixed adenosquamous carcinomas, and interestingly, bigenic tumors showed a phenotype resembling both oncogenes. All tumor types contained CK8-positive luminal epithelial cells. Interestingly, markers of myoepithelial (CK14) and progenitor (CK6) cells were only detected in mammary tumors arising from MMTV-CD8-IGF-IR mice and bigenic mice. This data is consistent with the notion that expression of IGF-IR in the mammary gland of transgenic mice may results in expansion myoepithelial cells expressing CK14 and progenitor cells expressing CK6 thus resulting in a mixed lineage differentiation in the tumor. We hypothesized that the difference in tumor histology may relate to the transformation of different progenitor cell types by IGF-IR and ErbB2. To investigate this, we have developed a mouse model system using avian RCAS retrovirus as vehicle to deliver CD8-IGF-IR or ErbB2 into mammary glands of transgenic mice expressing the avian receptor TVA under the control of a MMTV promoter. This allows retroviral infection of specific mammary cell types in vivo. Mammary intraductal injection of RCAS-CD8-IGF-IR into MMTV-TVA transgenic mice resulted in expansion of CK14 cells and disruption of normal mammary gland architecture. We are currently investigating the differences between CD8- IGF-IR and ErbB2-induced mammary tumorigenesis using this novel viral transduction system.
What is the impact of neutrophil elastase or plasma kallikrein processing of HGF in physiological or pathophysiological repair? During normal wound healing where the activities of proteases and their inhibitors are well balanced these events might present a physiological mechanism to protect epithelial or other cells from overwhelming HGF stimulatory effects. In contrast, in chronic inflammatory processes, as the non-healing wound microenvironment where proteases are significantly increased over protease inhibitors, a shift to the production of HGF fragments with attenuated or antagonistic HGF activities occurs, which impairs the healing response. Interestingly, in non-healing wounds HGF levels (or those of HGF cleavage products) were significantly increased over those in healing wounds and increased levels of antagonistic HGF fragments might result in a dominant negative effect, thus contributing to impaired HGF-mediated healing responses. Indeed, the attenuated signal of phosphorylated c-Met observed in non-healing versus healing wounds might at least in part be caused by lack of active HGF and/or antagonistic HGF fragments.
In order to investigate what happened to the tight junction during the biogenesis after the calcium switch, the amounts of tight junction proteins such as claudin-1 to –4; ZO-1 and ZO- 2; MUPP1 as well as the phosphorylated p44/42 ERK proteins in the cell were examined by Western blotting. A possible role of activated p44/42 ERK was examined because Singh & Harris (2004) have shown that the MAPK/ERK pathway is involved in the increase of TER in MDCK II cells upon the treatment with epidermal growthfactor (EGF). First samples of both the wild type MDCK II and the fusion receptor stable cell line at a later time point (23 hours) were examined. At this time point the total amount of claudin-2 was reduced several fold by activation of the fusion receptor whereas there were no noticeable changes in the amount of claudin-1, claudin-3, claudin-4, ZO-1, ZO-2 and MUPP1. Claudins constitute a family of transmembrane tight junction proteins which determine the tightness of the tight junction by the combination and mixing ratios of different claudin species. Out of the 24 family members, claudin-1 to –4 are known to be present in MDCK II cells. It has been demonstrated that the presence of claudin-2 could transform a high resistance (tight) MDCK I to a low resistance (leaky) MDCK II monolayer (Furuse et al., 2001). In addition, when claudin-4 was selectively removed from the tight junction the TER was significantly reduced (Sonoda et al., 1999). So, the presence of high amounts of claudin-2 and claudin-4 would correlate with the ‘loose’ and the ‘tight’ tight junction respectively. Thus, there is a good correlation of the agonist-induced high TER in the fusion receptor sample with a low amount of claudin-2 in the cell. At this point, it was tempting to speculate that the increase in TER observed in the agonist treated sample was due to selective degradation of claudin-2 induced by the agonist.
Therefore, the KRAS mutation is thought to lead to an increased ligand synthesis, which then establishes an autocrine loop to activate the EGFR. As a consequence, the PI3K-AKT pathway is activated and mediates pro-survival signals that can lead to radioresistance. It has been demonstrated that cancer cell lines carrying a mutant KRAS can be radiosensitized, while other cancer cell lines with the wild type KRAS protein showed no radiosensitizing effect when treated with an EGFR inhibitor (Toulany et al., 2007). These results require validation, because in these studies KRAS mutant NSCLC A549 cells were compared with the KRAS wild type pharyngeal squamous cell carcinoma cell-line FaDu (Toulany et al., 2007). Due to their different epithelial origins the two cell lines are expected to carry multiple additional genetic differences that could influence the effect of EGFR inhibition on their signaling networks.
We have so far only observed the upregulation of EGFR in MCF7 cells upon stable flotillin depletion. Since MCF7 cells display a constitutively active PI3K due to the E545K mutation , this prompted us to study if increased PI3K signaling might be the cause of EGFR upregulation upon flotillin-1 silencing. Indeed, EGFR amount was efficiently downregulated upon inhib- ition of PI3K activity. EGFR is not upregulated e.g. in human breast epithelial MCF10A, cervix carcinoma HeLa or human keratinocyte HaCat cells upon stable flotillin-1 knockdown (our unpublished findings). Ex- pression of flotillins in these cells lines is not much dif- ferent from MCF7 cells, but they all exhibit a WT PI3K . This may suggest that flotillins are required to keep EGFR amount under control when PI3K is constitutively activated. This is very likely to occur at least in part by means of increased activation of an as yet unidentified transcription factor that regulates EGFR transcription (see also above) and whose activation also depends on PI3K signaling. Since activating PI3K mutations that are oncogenic [11,12] are present in about 25% of breast tu- mors [7-9], and E545K is one of the most common PI3K mutations in breast cancer, it will be of uttermost im- portance to clarify the mutation status of breast cancer patients before aiming at treatments based on flotillin ablation.
Pulmonary arterial hypertension (PAH) constitutes a group of severe and progressive diseases characterized by oblitera- tion of pulmonary arteries leading to increased pulmonary vascular resistance [ 5 , 20 ]. A subsequent increase of right ventricular (RV) afterload leads to RV failure which is the leading cause of death in end-stage pulmonary hyperten- sion (PH) [ 35 ]. Although molecular mechanisms of disease remain poorly understood [ 45 ], early changes of endothelial cells (EC) appear to be crucial in the pathogenesis of a prolif- erative vasculopathy that represents the key histo-pathologic finding of PAH [ 11 , 42 , 43 ]. For example, Tuder et al. [ 50 ] have identified tumorlets of endothelial cells obliterating medium-sized arteries and suspected deregulated EC growth to drive the vasculopathy of human PH. Endothelial cells are the building blocks of vascular networks that enable oxygen and nutrient delivery throughout a tissue, but also serve as a rich resource of factors which maintain EC integrity in an autocrine fashion. Vascular endothelial growthfactor (VEGF) and its tyrosine kinase receptor, VEGF receptor-2 (VEGFR-2) play a central role in angiogenesis, endothelial cell protection, but also in the destabilization of endothe- lial barrier function. In vitro, specific inhibitors against VEGFR-2 [ 49 ], monoclonal antibodies directed against VEGFR-2 [ 26 ], and phenotypic knockout of VEGFR-2 are capable of inhibiting neoangiogenesis. In rodent models the inhibition of VEGF signaling by the tyrosine-kinase inhibitor Sugen5416 (SU5416) has been shown to aggravate vascular remodeling triggered by chronic hypoxia and to reproduce some of the angioproliferative features typical for advanced human PAH [ 44 , 48 ]. Concomitant administration of apoptosis inhibitors could prevent the effect of growthfactor inhibition, suggesting that loss of survival signals coupled with increased apoptosis lead to apoptosis-resist- ant ECs with abnormal growth potential [ 11 , 48 ]. Because these pre-clinical models and reports from large oncology registries suggest that angiogenesis inhibitors may induce PH, we aimed to dissect the consequences of interruption of VEGFR-2 signaling [ 17 , 29 , 33 ]. For this purpose, we investigated hemodynamic and histological effects of direct
CD44 v6 containing isoforms may sequester negatively-regulating elements from c-Met which is required for c-Met activation. There seems to exist such negatively-regulating elements. In constitutively active oncogenic Tpr-met (the extracellular domain of c-Met is replaced by Tpr sequences which provide two strong dimerization motifs), the juxtamembrane domain is lost which contains such a negative control region (Vigna et al., 1999). When this region was included into the fusion protein, Tpr-met dependent cell transformation was inhibited (Vigna et al., 1999). The negative control region could be a binding site for a phosphatase. It was reported that several protein tyrosine phosphatases (DEP-1, PTP-S and PTPase LAR) interacted with c-Met as a negative regulator (Palka et al., 2002, Villa-Moruzzi et al., 1998, Kulas et al., 1996). Interestingly, the receptor-tyrosine phosphatase DEP-1 dephoshorylated particular tyrosine residues (Tyr1349 and Tyr1365), indicating that it may function in controlling the specificity of signals induced by c-Met (Palka et al., 2002). There exist other negative regulators of c-Met, such as c-Cbl (Peschard et al., 2001, Taher et al., 2002) and ganglioside GD1a (Hyuga et al., 2001). c-Cbl binds also to the juxtamembrane domain of c-Met (Peschard et al., 2001).
More recently, the ML 1 gene was sequenced and its intron-free gene was cloned , and in a coassociation process from the recombinant A-chain and B-chain the non- glycosylated 57 kDa Viscumin heterodimer, rViscumin, was obtained . While the A- chain mediates the ribosome inactivation, the B-chain displays a carbohydrate binding activity which allows cellular uptake of the protein, and both activities of the molecule are needed for the observed cytotoxic effect of rViscumin on target cells . The recombinant drug rViscumin was studied extensively in preclinical models. It was found to possess antineoplastic and immunomodulatory properties in vitro and in vivo and was tolerated well in animals [284-287]. Furthermore, the potential receptor involved in the binding of rViscumin has been identified [288,289]. In contrast to another well-known ribosome- inactivating agent, ricin, rViscumin was therefore characterized as a sialic acid-specific type II ribosome-inactivating protein. Neolacto-series gangliosides with a Neu 5Acα2-6Galβ1- 4GlcNAc-terminus (CD 75s) were defined as the rViscumin receptors for the B-chain, leading to internalisation of the holoprotein .
this vasculopathy(16, 25). In the healthy vasculature, ECs form a monolayer above SMCs and act as a non-thrombogenic barrier for circulating macromolecules. ECs are responsible for oxygen and nutrient transfer to the underlying tissue(24). Additionally, ECs maintain the delicate balance between vasodilatation and vasoconstriction in a paracrine manner. Another mechanistic hypothesis of PAH is that there is an imbalance in vasoconstriction and vasodilatation of the pulmonary arteries. In alignment with all hypotheses, endothelial dysfunction and proliferation represents the integral pathologic finding in PAH 5-6 . Tuder et al have observed tumorlets of ECs to obliterate pulmonary arteries and these investigators suspected deregulated endothelial cell growth to underlie the vasculopathy of human pulmonary hypertensive disease. Drivers of monoclonal expansion of ECs with low turnover rate are poorly understood. Nevertheless endothelial injury (hypoxic assaults, toxins, autoimmune mechanisms, shear stress, infections) seems to be the initial step leading to endothelial cell dysfunction and apoptosis, which then initiate other mechanisms promoting the pathogenesis of PH 7 (26).
Even though FGFR3 is not highly expressed in CRC, the splice variant FGFR3-IIIc mediates FGF18-dependent survival signaling in colorectal tumors from the adenoma stage [10,13] . FGFR3-IIIc is upregulated in advanced CRC, and pathway blockade induced apoptosis  . Results of this study show that both endogenous expression and enforced expression of the receptor are related to low sensitivity of cells toward IRI. The IC50 concentrations are shifted by a factor of about 4 for IRI and 3 for SN38 ( Figure 3 ). This cannot be explained by increased cell growth in the overexpressing cultures because the impact on proliferation is moderate for FGFR3-IIIc 
Epithelial-to-Mesenchymal Transition (EMT) beschreibt einen hochdynamischen Prozess, bei dem Epithelzellen Differenzierungsmerkmale mesenchymaler Zellen entwickeln. Dies spielt eine entscheidende Rolle bei der Tumorprogression und vereinfacht die Metastasierung von Krebszellen. Als Grundlage von EMT sind strukturelle Veränderungen im Aktinzytoskelett notwendig, jedoch sind die verantwortlichen, aktinregulierenden Faktoren nur unzureichend charakterisiert. Formine stellen hierbei eine Proteinfamilie dar, die an zahlreichen, zellulären Prozessen, wie der Zytokinese, der Adhäsion und der Motilität beteiligt ist. Als Rho-GTPase- Effektoren formen Formine die größte Gruppe bekannter Aktinnukleatoren und neuerdings auch pharmakologisch relevante Ziele. In diesem Kontext wurde eine entscheidende Rolle von Formin-like 2 (FMNL2) bei der Assemblierung von Aktinstrukturen an de novo gebildeten Zell-Zell-Kontakten in 3D-Zellkulturmodellen beschrieben. Diese Aktivität entsteht abwärts von Rac1 sowie durch eine direkte Interaktion von FMNL2 und Komponenten des Cadherin-Catenin-Komplexes. Darüber hinaus wurde eine Phosphorylierung von FMNL2 durch PKC als essentiell für den zellulären Stoffwechseln von β1-Integrinen und damit für die Invasion von Krebszellen herausgestellt.
Following migration, both oligodendrocyte proliferation as well as differentiation can be supported by astrocytes. Ciliary neurotrophic factor (CNTF), e.g., promotes OPC survival 6 and maturation into a myelinating phenotype 181 . Furthermore, it is an upstream regulator of fibroblast growthfactor 2 ( FGF2) 6 which is known to be a mitogen for OPCs 19 . Further molecules which were attributed to be involved in oligodendrocyte differentiation are: Adenosine triphosphate (ATP) 81 , its degradation product adenosine 182 , insulin-like growthfactor 1 (IGF1) 65, 77 , interleukin 1 β (IL1β) 116 , interleukin 11 (IL11) 225 and leukemia inhibitory factor (LIF) 81 which were shown to directly (adenosine, IGF1, IL11, LIF) or indirectly (ATP, IL1 β) stimulate OPC differentiation and maturation. Additionally, transforming growthfactor β (TGFβ), in the presence of platelet-derived growthfactor (PDGF), also reduces proliferation and enhances differentiation in vitro 120 . On the other hand, TGF β promotes Jagged1 production in astrocytes which can bind to Notch1 on oligodendrocytes and retains them in a premature state 86 (figure 4, A). Even the role of the glial scar in remyelination is ambivalent. Mostly, OPCs are found at the rim of demyelinated lesions and remyelination can be supported by above-mentioned mechanisms. However, as astrogliosis might impair migration of OPCs, they are barely found in the lesion center 17, 37 .
receptor activation by forming disulfide-linked receptor dimers, leading to activation of the Ras/MAPK, STAT or PI3K pathway . FGFR3 mutations are reported in up to 50% of cancers of all stages from the lower and upper urinary tract with p.S249C being the most common mutation, found in 61% of cases . Mutation is inversely correlated with tumor stage and grade , and mutated tumors are associated with a favorable clinical outcome [8, 15, 20]. Despite the high mutation frequencies in urothelial carcinoma, the role of FGFR3 mutations in squamous differentiated bladder cancers is not well defined. In our study 6 of 71 (8.5%) tumor samples (n = 3 pure squamous carcinomas and n = 3 mixed carcinomas) contained an FGFR3 p.S249C mutation, all of them being grade 2 or higher and stage 3 or higher. This is slightly less than the frequency in invasive urothelial carcinomas reported by the TCGA project (12%)  and the n = 126 UC samples of Helsten et al. (15%) . Thus, we suggest that FGFR3 mutation (and FGFR3 mRNA expression) plays a minor role in squamous tumorigenesis. However, our survival analysis revealed an association between FGFR3 mutation and an
Zum GH-IGF-System gehören neben den oben erwähnten IGFs sechs IGF-Bindungsproteine (engl. insulin like growthfactor-binding proteins, IGFBPs) mit zugehörigen Proteasen sowie zwei IGF-Membranrezeptoren [8; 9]. Die IGFBPs fungieren als Trägerproteine, verlängern die Halbwertszeit des IGF-I und beeinflussen seine Verteilung in den verschiedenen Kompartimenten sowie die Affinität zu den Zellrezeptoren [4,8,10,11]. Darüber hinaus besitzen die IGFBPs eigene, IGF-I-unabhängige Wirkungen [4,12]. Unter den IGFBPs stellt das IGFBP-3 das Hauptbindungsprotein dar, welches zusammen mit IGF-I und der acid labile subunit (ALS) den sog. ternärer Komplex bildet [8,13]. Nur ein geringer Teil des IGF-I zirkuliert frei im Blut, der Großteil liegt in einem Komplex mit einem der IGFBPs vor [4,13]. Erst nach Abspaltung der IGFBPs durch Proteasen kann IGF-I an seine Zielrezeptoren binden und seine biologische Wirkung entfalten [4,9]. Aus diesem Grund wird bei der Messung des IGF-I zwischen dem totalen und dem freien, biologisch aktiven IGF-I differenziert. Letzteres kann über die Ermittlung des IGF-I/IGFBP-3 Verhältnisses abgeschätzt werden [4,11].
Veränderte Blutfettprofile sind ein Risikofaktor für die Entstehung kardiovaskulärer Erkrankungen und eine Komponente des metabolischen Syndroms. Vorangegangene Studien mit Patienten, welche von einer Störung der Wachstumshormon (GH) - Synthese betroffen sind, weisen auf einen Einfluss von Insulin like GrowthFactor-I (IGF-I) und IGF-Binding Protein-3 (IGFBP-3) auf den Fettstoffwechsel hin. Populationsbasierte Studien zeigten dabei widersprüchliche Ergebnisse. Ziel der Arbeit war die Analyse der querschnittlichen und längsschnittlichen Zusammenhänge zwischen IGF-I bzw. IGFBP-3 und Blutfetten (Gesamt-, Low density lipoprotein (LDL) -, High density lipoprotein (HDL) - Cholesterin, Triglyceride) in einer großangelegten Bevölkerungsstudie.
Unlike insulin, circulating IGFs are bound to six IGFBPs, which elicit at least two functions: prolongation of the half-life of circulating IGFs and neutralization of their metabolic effects. IGFBPs themselves are regulated by protease activity and some may have IGF-independent actions. IGFBPs compete with IGF receptors for IGF binding since they have a considerably higher affinity for IGFs than the IGF-I receptor (Kelley et al., 1996; Rajaram et al. 1997; Hwa et al., 1999). In the circulation, ~75-80% of the IGFs are present in a complex of ~150 kDa, a smaller proportion of 20-25% is associated in binary complexes, and less than 1% is found in a free form. The ~50 kDa binary complex is made up of several IGFBP species (IGFBP-1 to - 6) that are incompletely saturated with IGFs. The ~150 kDa complex is a ternary complex consisting of IGF-I or IGF-II plus IGFBP-3 or -5 and a non IGF binding component, the acid labile subunit (ALS) (Rajaram et al., 1997). In addition to the previously mentioned high- affinity binders, the existence of low affinity binders, such as IGFBP-related proteins and proteolysed IGFBPs is known. Contrary to IGFBPs, IGFBP-rPs and IGFBP fragments mediate biological effects mainly by IGF-independent mechanisms (Hwa et al., 1999).