Muc4, a heterodimeric transmembrane mucin containing EGF-like domains, has been described as an ErbB2-binding protein which modulates signaling via the ErbB2-ErbB3 pathway. In Muc4-transfected MCF-7 cells, Muc4 expression resulted in alteration of both the time course and phosphorylation levels of NRG beta 1 induced phosphorylation and activation of both ErbB2 and ErbB3. Muc4 significantly enhanced the autophosphorylation of ErbB2 over the early (defined 0-30 min) and intermediate (30-120 min) NRG beta 1 treatment times at three sites, Y1248, Y1221 and Y1139. The sites displayed differential maximal phosphorylation times. At Y1248 and Y1139, maximal phosphorylation occurred entirely during the early treatment phase. However, Y1221/2 showed maximal phosphorylation during the intermediate phase with a smaller peak during the early phase. The ratio of phosphorylated ErbB3 and total receptor level was significantly enhanced (in cells that expressed Muc4 compared without Muc4) over both the early and intermediate NRG beta 1 treatment time at the Y1289 site. This motif is one of several similar ErbB3 motifs whose phosphorylation mediates the binding of PI3-kinase. This phospholipid kinase is a key modulator of numerous cellular pathways leading to proliferation, motility and survival. Aberrancies in the ErbB2-ErbB3 signaling pathway have been implicated in the aggressive behavior of tumor cells, and the identification and characterization of modulators of this pathway are being sought as targets of potential therapeutic interventions. Muc4 significantly enhanced activated ERK in the absence of NRG beta 1 treatment while a NRG beta 1 mediated activation of AKT was observed. At early NRG beta 1 treatment time phases, Muc4 co-localized with phosphorylated ErbB2 (pY1248) independent of NRG beta 1 treatment; co-localization of Muc4 and ErbB2 receptor (activated/receptor forms) was observed at the apical surface or around the cell surface membrane. These data provide evidence in the Muc4-transfected MCF-7 cells for the biological NRG beta 1 mediated ErbB2 and ErbB3 activation. Our data suggests that Muc4 affects steady state phosphorylation levels and duration of the phosphorylation signal of both ErbB receptors, and that NRG beta 1 might affect ErbB2 and ErbB3 signaling differently. Additionally, the results of the timing of phosphorylation studies suggest the possibility that temporal aspects of phosphorylation at different sites may determine the pathways activated preferentially in the subsequent signaling cascades.

The pancreas is an endodermally derived organ consisting of three major cell lineages. The endocrine cells, organised into the Islets of Langerhans, regulate blood glucose homeostasis by producing and secreting hormones such as glucagon and insulin into the bloodstream. The major part of the pancreas consists however of acinar cells that produce digestive enzymes that are transported via a highly branched ductal system to the duodenum where they function in breakdown of food. Early in pancreas development a dorsal and ventral evagination of the foregut epithelium appear, resulting in the formation of the dorsal and ventral pancreatic bud. These pancreatic buds subsequently grow, branch and differentiate to form the mature pancreas via a process controlled by intrinsic factors, such as transcription factors, and extracellular signals. Insulin promoter factor 1 (Ipf1), also known as Pdx1 (for Pancreatic duodenal homeobox gene 1), is required for pancreas development. Although the evagination of pancreatic buds still occurs in Ipf1/Pdx1 mutant mice, the subsequent proliferation, branching and differentiation is impaired, resulting in complete pancreatic agenesis. Gene array profiling identified several candidate Ipf1/Pdx1 target genes, including FgfR2IIIb, ErbB3, Ptf1a/p48, Pax6 and Nkx6.1, in pancreatic progenitor cells. Together these genes provide a mechanistic explanation for the pancreatic growth arrest observed in Ipf1/Pdx1 deficient mice. In addition, Spondin1, which has not previously been described in the pancreas, was identified to be regulated by Ipf1/Pdx1. The spatial and temporal expression pattern of Spondin1 defines Spondin1 as a marker for early pancreatic progenitor cells. The Notch signalling pathway controls cell type specification and differentiation during pancreas development. The Fringe family of proteins have previously been shown to regulate Notch signalling by altering the interaction between Notch receptors and their ligands, hence affecting the cellular response. Manic Fringe (MFng) is transiently expressed in pancreatic pro-endocrine cells between E9.5 and E14.5. The expression of MFng is regulated by Ngn3, which may suggest a role for MFng in pro-endocrine cell maturation. The lack of a pancreatic phenotype in transgenic mice overexpressing MFng in the pancreatic epithelium and in MFng null mutant mice, however, provide evidence that MFng is dispensable for the specification, differentiation and function of the adult pancreas. Inhibitors of DNA binding (Id) proteins are generally known as inhibitors of differentiation, a feature they mainly perform by forming non-functional heterodimers with bHLH proteins, thereby inhibiting downstream targets of the bHLH proteins. Id proteins also promote cell proliferation by interacting with the cell cycle machinery. In the developing pancreas Id2 and Id3 are co-expressed in an overlapping manner during the period of massive proliferation and expansion of the pancreatic epithelium, suggestive of a role for the Id proteins during these processes. In addition, Id4 expression is also detected in the embryonic pancreas, albeit at lower levels. Gain- and loss- of- function analyses suggest however that specification, differentiation and function of the adult pancreas are largely independent of Id function.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

Pour croître et proliférer, les cellules cancéreuses de la prostate activent des voies de signalisation dépendantes des androgènes. L'intervention thérapeutique en première ligne du cancer de la prostate (CaP) s'appuie donc d’abord sur le blocage de l'axe androgènes-récepteur aux androgènes (RA) mais rapidement, les patients développent des tumeurs résistantes (CRPC, Castration Resistant Prostate Cancer).Les récepteurs à activité tyrosine kinase de la famille ErbB semblent jouer un rôle dans cette résistance, en particulier le récepteur ErbB3. En effet, l'inactivation des voies en aval d'ErbB1 et ErbB2, en association avec les anti-androgéniques n'empêche pas la progression vers l'hormono-indépendance, et une accumulation nucléaire d'ErbB3 est observée dans les CRPC en même temps que la voie PI3K-Akt est réactivée.Dans ce contexte, nous avons validé l'expression d'une isoforme nucléaire ErbB380kDa chez les patients et dans des lignées hormono-sensible (LNCaP) et hormono-résistante (PC3). Par ChIP-on-chip, nous avons isolé 353 promoteurs cibles communs aux deux lignées, 245 spécifiques à la lignée LNCaP et 925 à la lignée PC3, et montré qu'ErbB380kDa est un co-régulateur transcriptionnel des gènes étudiés, parmi lesquels GATA2. L'analyse in silico de ces promoteurs révèle des sites de liaison pour les facteurs de transcription GATA2 et MZF1 au niveau des régions liant ErbB380kDa. Un complexe nucléaire GATA2-MZF1-ErbB380kDa est retrouvé dans les cellules LNCaP et PC3.Des travaux récents montrent que GATA2 s'associe au RA pour réguler l'expression de gènes et qu'il pourrait être participer à la dissémination métastatique dans le CaP.Nos résultats suggèrent qu'ErbB380kDa pourrait jouer un rôle régulateur, en amont de GATA2, dans les processus de résistance et l'apparition de métastases. Cette isoforme nucléaire insensible aux traitements actuels apparaît donc comme une cible privilégiée pour le ciblage thérapeutique. / Prostate cancer (PCa) is dependent on androgens and functional androgen-receptor (AR) for growth and proliferation. Androgen-directed therapy is used at the first stages of the disease but cancer cells frequently become resistant (CRPC) by inappropriate reactivation of AR activity. As ErbB receptors are expressed in PCa cells, therapies aiming at inactivate the pathways downstream have been tested in advanced prostate cancers alongside hormone-based therapy. Still, a significant proportion of CRPC treated by ErbB1/2 inhibitors resist to treatment. ErbB3 could be responsible for this failure through both its unexpected nuclear localization and the reactivation of the PI3K-Akt pathway in those advanced tumors.We have described a nuclear ErbB380kDa isoform, expressed in hormone-sensitive (LNCaP) and hormone-resistant (PC3) PCa cell lines that accumulates in the nucleus of tumor cells during cancer progression. ChIP-on-chip experiments led us to characterize 353 target promoters binding ErbB380kDa in both cell lines; 245 promoters specific to LNCaP and 925 specific to PC3 cells, among which the promoter of GATA2. We show that ErbB380kDa functions as a transcriptional co-regulator for the studied genes, potentially through its interaction with transcription factors. In silico analysis revealed binding sites for GATA2 and MZF1 transcription factors on the target promoters, and a complex GATA2-MZF1-ErbB380kDa has been found in LNCaP and PC3 cells. Recent publications have reported a role for GATA2 in the regulation of RA responsive-genes and in metastatic spreading. We propose that ErbB380kDa could act, upstream of GATA2, to induce resistance mechanisms and facilitate cancer progression. Thus, ErbB380kDa emerges as a putative target for the development of new therapies in prostate cancer.

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

Prostate cancer is the most common cancer affecting men today. Therefore, there is a strong need for accurate biomarkers and successful therapeutic treatments. A novel approach combining a computationally built protein-protein interaction network of proven microRNA protein targets with high throughput proteomics identified ErbB2 and ErbB3 as key proteins in prostate cancer. These results coupled with microRNA array screening of an androgen-independent prostate cancer progression model, substantiated by single microRNA analysis, suggested miR125b as a key tumor suppressor contributing to prostate cancer progression. miR125b expression was shown to be substantially increased in the non-tumorigenic P69 cell line compared to its highly tumorigenic, metastatic M12 variant. Luciferase reporter gene assays including the entire 3’UTR of either ErbB2 or ErbB3 revealed a 2.8- and 2.4-fold decrease (respectively) compared to control vector. Thus, this combinatorial approach has suggested an additional microRNA and its target involved in prostate tumor progression.

Primary brain tumours, and especially the most common form malignant gliomas, usually display a pronounced resistance to other treatment modalities when surgery fails to cure. Growth factors, such as EGF and its receptor, frequently amplified and overexpressed in malignant gliomas, and factors associated with multidrug resistance have been suggested to at least partially explain the poor outcome. The aim of this thesis was to characterise factors in primary brain tumours associated with the development of resistance with focus on the epidermal growth factor receptor (ErbB) family, and multidrug resistance (MDR). Influences of irradiation on the expression and activity of P-glycoprotein (Pgp) in malignant gliomas was evaluated. The effects showed that irradiation increased the efflux activity of Pgp in rat brain vascular endothelial cells, but not in glioma cells. In the intracranial BT4C glioma model, Pgp was detected in the capillary endothelium in the tumour tissue but not in glioma cells. Expression of several factors coupled to MDR (Pgp, MRP1, LRP, and MGMT) in primary brain tumours were analysed and correlated to clinical data. In gliomas, Pgp and MRP1 were predominantly observed in capillary endothelium and in scattered tumour cells, whereas LRP occurred only in tumour cells. In meningiomas, expression of the analysed markers was demonstrated in the capillary endothelium, with a higher expression of Pgp and MRP1 in transitional compared to meningothelial meningiomas. A pronounced expression of MGMT was found independently of the histopathological grade or tumour type. Survival analysis indicated a shorter overall survival for patients suffering from low-grade gliomas with high expression of Pgp. To explore the importance of the epidermal growth factor receptor (EGFR), expression levels of the family members (EGFR, ErbB2-4) were analysed and their relations to various clinical parameters were evaluated in gliomas and meningiomas. In gliomas, the highest EGFR expression was observed in high-grade tumours, while ErbB4 expression was most pronounced in low-grade tumours. In meningiomas, expression of EGFR, ErbB2, and ErbB4 was observed in the majority of the tumours. An intriguing observation in low-grade gliomas was a significantly decreased overall survival for patients with high EGFR protein expression. The effects of different time schedules for administration of the selective EGFR inhibitor ZD1839 in relation to irradiation of glioma cells were analysed. The analyses showed a heterogeneity in the cytotoxic effects of ZD1839 between cell lines, and it was obvious that some of the cell lines showed sensitivity to ZD1839 despite no or low expression of EGFR. The study also demonstrated the importance of timing of ZD1839 administration when this agent is combined with irradiation. In conclusion, in order to enhance the efficacy of radiotherapy by various drugs in malignant gliomas it may be essential to inhibit drug efflux activity in endothelial cells and to deliver drugs in an optimal timing in relation to radiotherapy. The heterogeneity in expression of drug resistance markers, as well as the ErbB family reflects the complexity in classification of primary brain tumours, and indicates that subgroups of patients with low-grade gliomas expressing Pgp and EGFR might benefit from more aggressive and individualised treatment.

No description available.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3. / <p>QC 20170330</p>