Sperm protein, enterokinase and agrin (SEA) modules are small folds within large heavily glycosylated modular proteins. Because decreased expression of SEA-containing proteins such as perlecan (PLN) can lead to diseases such as Schwartz-Jampel syndrome (SJS), characteristics of the PLN SEA module including folding, potential for autocleavage, and protein binding were studied. Sequence analyses, recombinant protein evaluation, and a yeast two-hybrid screen were used to study the PLN SEA module and compare it to the mucin (MUC) 1 SEA module. In silico modeling of the PLN SEA module demonstrated a well conserved α/β sandwich fold. Experiments with expressed proteins showed that unlike MUC1, the PLN SEA module does not autocleave. Two-hybrid screening identified four “high confidence” proteins as potential binding partners which were explored in preliminary experiments. Together, these results demonstrate that PLN SEA module is unique and its properties cannot be generalized with other SEA module proteins such as MUC1.

The epidermal growth factor receptor (EGFR) belongs to the erbB family of receptor tyrosine kinases which consists of four members (EGFR, ErbB2, ErbB3 and ErbB4). Upon ligand binding, the EGFR is capable of dimerization with other erbB receptors and propagates signals regulating a diverse array of cellular physiologies, including cell growth, migration and survival. Dysregulation of the EGFR is important for development and progression of different types of cancers, including breast cancer. Breast cancer is the second leading cause of cancer death in women. EGFR overexpression has been observed in about 15% of all breast cancers. Moreover, in triple negative breast cancer (TNBC), which is a more aggressive type of breast cancer and lacks effective therapies, up to 50% of tumors are found to overexpress EGFR. Targeted therapy against EGFR has been used in TNBC. However, limited efficacy has been observed in TNBC due to intrinsic and acquired resistant mechanisms. In order to overcome this issue, we have developed two novel therapeutic peptides derived from the nuclear localization signal (NLS) sequence and juxtamembrane domain of EGFR and investigated their efficacy in regard to inhibiting EGFR translocation and activation in TNBC. EGFR has been found to translocate into the nucleus and nuclear EGFR can affect gene transcription, cell proliferation, stress response and DNA repair through interacting with different components in the nucleus. Importantly, these functions of nuclear EGFR correlate with cancer prognosis and therapeutic resistance. We found that an EGFR NLS-derived peptide (ENLS peptide) could inhibit activated EGFR (pY845) undergoing nuclear translocation. We also showed that this ENLS peptide sensitized breast cancer cells to AG1478 (EGFR tyrosine kinase inhibitor) treatment. The juxtamembrane domain of EGFR regulates its trafficking to the nucleus and mitochondria, interaction with calmodulin and calcium signaling, and participates in dimerization and activation of EGFR. These non-traditional kinase related functions of EGFR represent a novel target for EGFR therapy. We found that a mimetic peptide of the juxtamembrane domain of EGFR (EJ1 peptide) could effectively inhibit EGFR activation through promoting inactive dimer formation. It could also effectively kill cancer cells through processes of apoptosis and necrosis. Mechanistically, this EJ1 peptide affects membrane integrity thereby leading to calcium influx, disruption of mitochondrial membrane potential and reactive oxygen species (ROS) accumulation. Importantly, EJ1 peptide appeared to be effective in inhibition of tumor growth and metastasis in a transgenic mouse model of breast cancer and showed no observable toxicity. ErbB3, another member of the erbB family, represents an important driver of the parallel signaling pathway to EGFR as well as a key regulator of PI3K/AKT activity which is important for therapeutic resistance. ErbB3 has been shown to interact with MUC1. The interaction between MUC1 and EGFR promotes EGFR stability through recycling of receptors. We found that MUC1 expression also affected ErbB3 activity and stability through ErbB3/EGFR/MUC1 complex formation. In conclusion, we demonstrated that two EGFR-derived peptides, working through novel strategies, represent a new foundation of effective therapeutic agents to breast cancer. ErbB3/EGFR/MUC1 complex formation under MUC1 expression also represents a druggable target for ErbB3 activity and stability.

The relationship between MUC1 and EGFR has been characterized by our lab to be highly tumorigenic. A peptide therapeutic was developed in our lab to block the cytoplasmic interaction of MUC1 and EGFR by competing with the EGFR-binding domain of MUC1. The peptide, PMIP, reduced invasion and proliferation in vitro and reduced tumor growth and metastasis in vivo. These studies demonstrated the potency of MUC1/EGFR interactions in tumor progression, and we sought to explore this concept further. We wanted to clarify a mechanism by which MUC1 and EGFR together drive breast cancer metastasis, and we identified c-Met as a mediator of MUC1 and EGFR-driven cell motility. In two separate assays, we demonstrated that c-Met activity was necessary for MUC1 and EGFR to promote migration and invasion. In addition, we wanted to identify the role of EGFR membrane localization in membrane identity and tumor initiation. We established several EGFR localization mutants to compare to wild-type basolateral EGFR and we performed proof-of-concept experiments to show that these mutants will be useful in future studies. Finally, we studied the effect of MUC1 and EGF loss on tissue architecture and function in the lactating mammary gland. EGF is the primary ligand for EGFR during lactation, and MUC1 is highly expressed during this period of mammary development. In addition, it has been shown that EGFR and MUC1 interact at the apical cell surface of lactating mammary ducts, yet there is no link between lactation and tumor formation. We hypothesized that MUC1 and EGFR interaction may have a role in maintaining tissue architecture and lactation function in the mouse mammary gland. We found instead that the loss of MUC1 and EGF had no noticeable effect on lactation and did not result in tissue defects. These studies further clarified the relationship between MUC1 and EGFR in several different contexts, showing a role for their interaction in metastatic progression, and showing that their ablation has no effect in the lactating mammary gland. Future studies will elucidate the role of MUC1 and EGFR interaction in tumor initiation, and we have taken several steps in our studies toward that goal.

EGF receptors are key regulators of cell survival and growth in normal and transformed tissues. Ligand binding results in formation of homo/hetero dimers of these receptors, followed by activation of the kinase activity and subsequent tyrosine phosphorylation of many downstream molecules. The activation of these receptors is not only mediated by the binding of their cognate ligands, but by transactivaton by other molecules as well. Recent studies have identified an oncogenic glycoprotein MUC1 as a binding partner for EGFR and that MUC1 expression can potentiate EGFR-dependent signal transduction. After receptor activation, EGFR is typically downregulated via an endocytic pathway that results in receptor degradation or recycling. We report here that MUC1 expression inhibits the degradation of ligand-activated erbB1. In addition, MUC1 expression results in prolonged activation of Akt, but not ERK1,2 MAPKinase. The MUC1-mediated protection against degradation occurs with a decrease in EGF-stimulated ubiquitination of erbB1, and an increase in erbB1 recycling. We then utilized the WAP-TGFα transgenic mouse model of breast cancer and determined that a loss of Muc1 expression dramatically alters mammary tumor progression. While 100% of WAP-TGFα/Muc1^(+/+) mice form mammary gland tumors, only 37% of WAP-TGFα/Muc1^(-/-) form tumors. Furthermore, expression of cyclin D1 expression is significantly suppressed in tumors derived from WAPTGFα/Muc1^(-/-) animals, and loss of Muc1 expression resulted in a significant inhibition in the formation of hyperplastic lesions in the mammary gland. We also observed metastatic pulmonary adenocarcinoma (1/29) and perivascular lymphoma of unknown origin (28/29) in the WAP-TGFα transgenic mice but not in the WAP TGFα/Muc1^(-/-) animals. To determine the effects of Muc1 expression on metastasis in a model lacking perivascular lymphoma, we crossed MMTV-Wnt-1 and MMTV-MUC1 transgenic mice and evaluated interactions between Muc1 and EGFR. Although the MMTV-Wnt-1 mice are non-metastatic, a majority (6/10) of the bitransgenic MMTVWnt- 1/MMTV-MUC1 formed pulmonary metastases. Furthermore, overexpression of MUC1 increases the breast cancer cell invasion in vitro. The MUC1 induced increase in invasion is found to be EGF and EGFR-kinase dependent. Collectively, these data indicate that MUC1 expression contributes to many of the hallmarks of cancer and in addition, is an important modulator of EGFR-associated mammary tumor progression.

In der vorliegenden Arbeit wurden Tumorimpfstoffe auf der Basis des Plasmid-Vektors pCI, modified vaccinia virus Ankara (MVA) und MVA-infizierten dendritischen Zellen entwickelt und durch Sequenzierung, Western blotting und durchflußzytometrische Analyse überprüft. Die in vivo Wirksamkeit der Vakzinen wurde in verschiedenen Tumormodellen in C57BL/6 Mäusen verglichen. Die auf dem eukaryotischen Expressionsvektor pCI basierende DNA-Vakzinierung induzierte einen sehr wirksamen, antigenspezifischen und langfristigen Schutz vor Muzin, CEA oder beta-Galactosidase exprimierenden Tumoren. Eine MVA-Vakzinierung bietet in den in dieser Arbeit durchgeführten Tumormodellen keinen signifikanten Schutz vor Muzin oder beta-Galactosidase exprimierenden Tumoren. <br /> <br /> Sowohl humane, als auch murine in vitro generierte dendritische Zellen lassen sich mit MVA &ndash; im Vergleich zu anderen viralen Vektoren &ndash; sehr gut infizieren. Die Expressionsrate der eingefügten Gene ist aber gering im Vergleich zur Expression in permissiven Wirtszellen des Virus (embryonale Hühnerfibroblasten). Es konnte gezeigt werden, daß eine MVA-Infektion dendritischer Zellen ähnliche Auswirkungen auf den Reifezustand humaner und muriner dendritischer Zellen hat, wie eine Infektion mit replikationskompetenten Vakzinia-Stämmen, und außerdem die Hochregulation von CD40 während der terminalen Reifung von murinen dendritischen Zellen inhibiert wird. Die während der langfristigen in vitro Kultur auf CEF-Zellen entstandenen Deletionen im MVA Genom führten zu einer starken Attenuierung und dem Verlust einiger Gene, die immunmodulatorische Proteine kodieren, jedoch nicht zu einer Verminderung des zytopathischen Effekts in dendritischen Zellen. <br /> <br /> Die geringe Expressionsrate und die beobachtete Inhibition der Expression kostimulatorischer Moleküle auf dendritischen Zellen kann für eine wenig effektive Induktion einer Immunantwort in MVA vakzinierten Tieren durch cross priming oder die direkte Infektion antigenpräsentierender Zellen verantwortlich sein.<br /> <br /> Durch die Modifikation einer Methode zur intrazellulären IFN-gamma Färbung konnten in vakzinierten Mäusen tumorantigenspezifische CTL sensitiv und quantitativ detektiert werden. Die so bestimmte CTL-Frequenz, nicht jedoch die humorale Antwort, korrelierte mit der in vivo Wirksamkeit der verschiedenen Vakzinen: DNA vakzinierte Tiere entwickeln starke tumorantigenspezifische CTL-Antworten, wohingegen in MVA-vakzinierten Tieren überwiegend gegen virale Epitope gerichtete CD4 und CD8-T-Zellen detektiert wurden.<br /> <br /> Die Wirksamkeit der pCI-DNA-Vakzine spricht für die Weiterentwicklung in weiteren präklinischen Mausmodellen, beispielsweise unter Verwendung von MUC1 oder HLA-A2 transgenen Mäusen. Die Methoden zur Detektion Tumorantigen-spezifischer CTL in 96-Loch-Mikrotiterplatten können dabei zur systematischen Suche nach im Menschen immundominanten T-Zell-Epitopen im Muzin-Molekül genutzt werden. <br /> <br /> Der durchgeführte Vergleich der auf den Vektoren pCI und MVA basierenden Vakzinen und die Analyse neuerer Publikationen führen zu dem Ergebniss, daß vor allem DNA-Vakzinen in Zukunft eine wichtige Rolle bei der Entwicklung von aktiven Tumorimpfstoffen spielen werden. Rekombinante MVA-Viren, eventuell in Kombination mit DNA- oder anderen Vektoren, haben sich dagegen in zahlreichen Studien als wirksame Impfstoffe zur Kontrolle von durch Pathogene hervorgerufenen Infektionserkrankungen erwiesen. / In this study, tumor vaccines based on the plasmid pCI, the attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) and MVA-infected dendritic cells were constructed and characterized by sequencing, Western blot and flow cytometric analysis. The efficiency to induce tumor immunity in vivo was compared in several C57BL/6 mouse tumor models. Naked DNA Vaccination based on the eukaryotic expression vector pCI did induce very effective, antigen-specific and long-term protection against tumor cell lines expressing mucin, CEA or beta-Gal whereas MVA vaccination did not elicit protective immunity against Mucin or beta-Gal expressing tumors. MVA does infect human or murine in vitro generated dendritic cells very efficiently compared to other viral vectors, however expression levels of the inserted antigens in dendritic cells are significantly lower than in permissive host cells (chicken embryo fibroblasts). <br /> <br /> It could be shown that the effect of MVA infection on the maturation status of dendritic cells is similar to the effects described for dendritic cells infected with replication competent vaccinia strains. In addition it was shown that the upregulation of the important costimulatory molecule CD40 through LPS stimulation is strongly inhibited in MVA infected cells. During passage in tissue culture, MVA has accumulated a number of large deletions, including a number of immunomodulatory molecules and resulting in a strong attenuation. However the strong cytopathic effect on dendritic cells is maintained. <br /> <br /> The low level of expression and the effect on dendritic cell maturation may be responsible for the failure of MVA to induce tumor immunity through either cross presentation or direct infection of antigen presenting cells.<br /> <br /> To detect and quantify tumor-antigen-specific CTL a method based on intracellular IFN-gamma staining was modified and it could be shown that the cellular &ndash; but not the humoral &ndash; response does correlate with in vivo protection: DNA but not MVA vaccines do induce high levels of tumorantigen-specific CTL whereas MVA-vaccines do induce strong and long lasting CD4 and CD8-T-cell responses against vaccinia antigens. <br /> <br /> The excellent protection induced by pCI-DNA-vaccination in different tumor models does encourage us to further investigate the elicitation of tumor immunity in MUC1 or HLA-A2 transgenic mice. In mice transgenic for human MHC-I, the IFN-gamma staining protocol could be used to systematically screen for mucin T-cell epitopes that are relevant in humans.

Le rôle de la salive dans la perception sensorielle est de plus en plus reconnu, notamment par le biais des interactions physico-chimiques pouvant s’établir entre protéines salivaires et constituants alimentaires. Ce travail s’intéresse à la pellicule salivaire, la couche de protéines salivaires ancrées aux cellules épithéliales, et vise à caractériser les interactions pouvant s’établir d’une part entre ces protéines et épithélium oral, et d‘autre part entre ces protéines et les molécules de la flaveur. Pour cela, un modèle in vitro de muqueuse orale a été développé. Une lignée cellulaire stable (TR146/MUC1) a été obtenue par transfection de la lignée cellulaire TR146 de manière à exprimer la mucine membranaire MUC1. Afin de former une pellicule salivaire, les cellules confluentes ont été incubées avec de la salive humaine. La rétention des mucines salivaires MUC5B par les cellules TR146/MUC1 est augmentée par rapport aux TR146, apportant ainsi un argument en faveur de l’implication de MUC1 dans l’ancrage des MUC5B aux cellules épithéliales. Le modèle développé a été appliqué à l’étude des interactions entre la muqueuse orale et les molécules d’arôme et les tanins. L’analyse des coefficients de partage par GC-FID a mis en évidence 1- l’importance de l’hydratation de la muqueuse sur la libération des composés les plus hydrophiles, 2- la capacité des cellules à métaboliser certaines molécules d’arôme, 3- l’absence d’effet de la pellicule sur la libération des molécules d’arôme à l’équilibre. En revanche, l’analyse par PTR-MS a révélé un effet de la muqueuse et de la pellicule sur la cinétique de libération des molécules d’arôme. Les interactions entre les protéines de la pellicule salivaire et les tanins modifient les caractéristiques structurales de la pellicule, en particulier le tapissage des cellules par les MUC5B. Les possibles implications sensorielles, respectivement dans les phénomènes de persistance aromatique et d’astringence, sont discutées. / The role of saliva in food sensory perception is increasingly recognized, especially through physicochemical interactions occurring between salivary proteins and food components. This work focuses on the mucosal pellicle, a layer of salivary proteins anchored onto epithelial cells, and aims at characterizing interactions that may occur between the proteins of the mucosal pellicle and flavour compounds. For that purpose, an in vitro model of oral mucosa was developed. A stable cell line (TR146/MUC1) was obtaining by transfecting the TR146 cell line in order to express the membrane bound mucin MUC1. In order to form a salivary pellicle, confluent cells were incubated with human saliva. A higher retention of salivary MUC5B by TR146/MUC1 cells was observed compared to TR146 cells, emphasising the involvement of MUC1 in MUC5B anchoring to epithelial cells. The model was applied to the investigation of interactions between the oral mucosa and aroma molecules and tannins. Measurements of partition coefficients by GC-FID revealed 1- the role of hydration of the mucosa on the release of the most hydrophilic compounds, 2- the ability of cells to metabolize some aroma compounds, 3- the absence of effect of the mucosal pellicle itself on aroma release at the thermodynamic equilibrium. Oppositely, analyses by PTR-MS evidenced an effect of the mucosa and of the pellicle on aroma release kinetic. Interactions between proteins of the mucosal pellicle and tannins modified structural characteristics of the pellicle, especially the coating of cells by salivary MUC5B. Sensory relevance for the phenomena of aroma persistence and astringency, respectively, are discussed.

El mal pronòstic en una neoplàsia epitelial està associada a l'adquisició de característiques mòbils o invasives per part de les cèl·lules canceroses. Aquesta transformació morfològica es denomina transició epiteli-mesènquima (TEM). Snail és un factor de transcripció implicat en aquest procés, responsable de reprimir l'expressió de l'E-cadherina. Aquest treball demostra que Snail té la capacitat de reprimir l'expressió de MUC1 i VDR a través de la seva unió directa a caixes de reconeixement situades en els diferents promotors proximals. A més, la sobreexpressió d'Snail en diverses línies cel·lulars provoca un augment dels nivells d'ARNm de ZEB1 i un increment de l'activitat del seu promotor. L'activitat del promotor mínim d'Snail i els seus nivells d'ARNm depenen de la senyalització d'ERK. Finalment, hem demostrat que Snail i WT1, un regulador positiu de l'expressió de l'E-cadherina, competeixen per unir-se al promotor de l'E-cadherina i regular la seva transcripció. / The poor prognosis in epithelial neoplasia is associated with the acquisition of motile or invasive properties by the cancerous cells. This morphological transformation is often referred to as epithelial to mesenchymal transition (EMT). The Snail transcription factor is involved in this process by repressing the expression of E-cadherin. In this study we demonstrate the capacity of Snail to repress both MUC1 and VDR transcription by direct binding to specific sequences within their proximal promoter. Moreover, Snail overexpression in several cell lines induces ZEB1 mRNA and increases its promoter activity. The activity of the Snail minimal promoter is dependent on the ERK signaling pathway. Finally, we have demonstrated that Snail and WT1, a positive regulator of E-cadherin expression, compete for the binding to the E-cadherin promoter in order to regulate its transcription.

No description available.

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1-&gt;3)GalNAcα-Ser/Thr) and Tn (GalNAcα-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases. Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''-&gt;2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine. Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency. A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes. The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.