Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. / <p>QC 20121122</p>

The aryl hydrocarbon receptor (AhR) binds synthetic and chemoprotective phytochemicals, and research in this laboratory has developed selective AhR modulators (SAhRMs) for treatment of breast cancer. Activation of the AhR through agonists such as TCDD inhibits hormone activation of several E2-responsive genes in breast cancer cell lines. In this study, inhibition of E2-induced proliferation and gene expression by TCDD has been investigated in the uterus of wildtype, ERKO and AhRKO mice. Cyclin D1, DNA polymerase ?, and VEGF mRNA levels are induced by E2 through ER? in the uterus as determined by in situ hybridization studies. TCDD down-regulated E2-induced cyclin D1 and DNA polymerase ? expression, but not E2-induced VEGF expression, in wild-type mice, but not AhRKO mice, confirming the role of the AhR. Furthermore, protein synthesis was not necessary for induction of cyclin D1 or DNA polymerase ?gene expression by E2 or inhibition of these responses by TCDD. Therefore, AhR-ER? crosstalk directly regulates the expression of genes involved in cell proliferation in vivo. AhR agonists induce down-regulation of ErbB family receptors in multiple tissues/organs suggesting possible inhibitory interactions with chemotherapeutic potential. Recently, it has been reported that the SAhRM 1,1??,2,2??-tetramethyldiindolylmethane inhibited DMBA-induced mammary tumor growth in rats and also inhibited MAPK and PI3-K pathways in human breast cancer cells. BT-474 and MDA-MB-453 cell lines are ErbB2-overexpressing breast cancer cells that express functional AhR and exhibit constitutive activation of MAPK and PI3-K pathways. Therefore, 1,1??,2,2??-tetramethyldiindolylmethane-induced inhibition of ErbB2 signaling was investigated in these cells lines and in the MMTV-c-neu mouse mammary tumor model, which overexpresses ErbB2 in the mammary gland. The growth of ErbB2 overexpressing cell lines and mammary tumors was inhibited by 1,1??,2,2??-tetramethyldiindolylmethane; however, modulation of MAPK or PI3-K pathways and cell cycle proteins nor induction of apoptosis by 1,1',2,2'-tetramethyldiindolylmethane was observed in the ErbB2overexpressing cell lines. Current studies are investigating mitochondrial effects of 1,1??,2,2??-tetramethyldiindolylmethane in the ErbB2-overexpressing cell lines, as well as continuing studies on gene expression profiles in the mammary glands of MMTV-c-neu mice to better understand and identify critical genes that are responsible for ErbB2-mediated transformation and growth of cancer cells/tumors.

The broad objective of this research is to examine the relationship between the cellular micromechanical environment and disease progression in cancer. The mechanical stiffness of cancerous tissue is a key feature that distinguishes it from normal tissue and thus facilitates its detection clinically. While numerous inroads have been achieved toward elucidating molecular mechanisms that underlie diseases such as cancer, quantitative characterization of associated cellular mechanical properties and biophysical attributes remains largely incomplete. To this end, the present research provides insight into the following questions: (1) What is the effect of extracellular matrix (ECM) stiffness and architecture on internal cancer cell rheology and cytoskeletal organization? (2) What are the integrated effects of ECM stiffness and cell metastatic potential on the intracellular rheology and morphology of breast cancer cells? (3) What are the integrated effects of ECM stiffness, ECM architecture, and cell metastatic potential on the motility of breast cancer cells? To examine these phenomena, the present research utilizes a multidisciplinary engineering approach that integrates experimental rheology, theoretical mechanics, confocal microscopy, computational algorithms, and experimental cell biology. Briefly, genetically altered cancer-mimicking cells are cultured within synthetic ECMs of varying mechanical stiffness and structure, where they are then observed using time-lapsed confocal microscopy. Image analyses and computational algorithms are then employed to extract measures of cell migration speed and intracellular stiffness via particle-tracking microrheology techniques. Major results show that ECM stiffness elicits an intracellular mechanical response only within the framework of physiologically relevant matrix environments and that a key cell-matrix attachment protein (the integrin) plays an essential role in this phenomenon. Additional results indicate that a well-known breast cancer-associated biomarker (ErbB2) is responsible for sensitizing mammary cells to ECM stiffness. Finally, results also show that a switch in ECM architecture significantly hinders the migratory capacity of ErbB2-associated cells, which may explain why the ErbB2 biomarker is detected with much higher frequency in early stage breast cancer than in later stage invasive and metastatic cancers. In total, these findings inform the fields of mechanobiology and cancer biology by systematically linking cell rheology, cell motility, matrix mechanics, and disease progression in cancer. / text

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19260号 / 理博第4115号 / 新制||理||1592(附属図書館) / 32262 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

La régulation de la transcription des gènes par les récepteurs des estrogènes ERα et ERβ joue un rôle important dans la croissance cellulaire et dans le développement du cancer du sein. Une augmentation de l’expression de CXCR4 et de son ligand SDF-1/CXCL12 corrèle avec un phénotype plus agressif du cancer du sein. Ici, nous démontrons un mécanisme de boucle de régulation positive entre la signalisation de CXCR4/SDF-1 et l’activité transcriptionnelle des ERs dans des cellules cancéreuses mammaires. L’activité transcriptionnelle de ER et l’expression de gènes cibles de ER, dont SDF-1 lui-même, sont augmentées dans la lignée cancéreuse mammaire MCF-7 en réponse à SDF-1. Ces effets sont bloqués par l’anti-estrogène fulvestrant et par la délétion de CXCR4. Par ailleurs, l’expression des gènes et la prolifération des cellules cancéreuses mammaires MCF-7 en réponse à l’estrogène sont altérées par l’inhibition de CXCR4. La signalisation par les facteurs de croissance joue un rôle important dans le cancer du sein. La surexpression et la dérégulation de la signalisation par le récepteur à activité tyrosine kinase ErbB2 corrèlent avec un phénotype tumoral mammaire plus agressif et un moins bon pronostic. Cependant, comment la signalisation de ErbB2 et de CXCR4 sont fonctionnellement reliées dans la régulation de la réponse de ER dans les cellules cancéreuses mammaires n’est pas connue. Nous démontrons ici que CXCR4 régule négativement l’expression protéique de ErbB2 et de son partenaire d’interaction ErbB3 ainsi que la phosphorylation de ErbB2. CXCR4 altère l’activation de la voie PI3-K/Akt par le dimère ErbB2/ErbB3 en réponse à héréguline alors qu’en présence de SDF-1, les niveaux d’activation sont récupérés. Nous avons trouvé que héréguline-β promouvoit la phosphorylation de la sérine 339 de CXCR4, un site important pour l’internalisation et la signalisation du récepteur. De plus, le recrutement de ErbB2 à CXCR4 est favorisé par ErbB3 et héréguline-β. L’activité transcriptionnelle ainsi que l’expression des gènes cibles de ER en réponse à l’héréguline sont relevées avec l’expression de CXCR4 et partiellement récupérées avec l’addition de SDF-1. Ces résultats démontrent que le recrutement de CXCR4 à ErbB2 altère la signalisation médiée par ErbB2/ErbB3 ainsi que l’activité hormonale de ER dans des cellules cancéreuses mammaires. Nous travaux ont permis d’identifier et de caractériser l’impact de la signalisation médiée par des récepteurs membranaires sur la réponse transcriptionnelle de ER dans des cellules cancéreuses mammaires. La signalisation membranaire est un facteur pouvant contribuer à la résistance aux thérapies endocriniennes et donc cibler les récepteurs impliqués s’avèrerait utile pour améliorer les traitements existants et mettre au point de nouvelles approches. / Induction of estrogen-regulated gene transcription by estrogen receptors ERα and ERβ plays an important role in breast cancer development and growth. High expression of the chemokine receptor CXCR4 and its ligand CXCL12/SDF-1 has also been correlated with aggressive breast tumor phenotypes. Here, we describe a positive regulatory loop between CXCR4/SDF-1 signaling pathway and ER transcriptional competence in human breast cancer cells. Treatment of breast carcinoma MCF-7 cells with SDF-1 increased ER transcriptional activity and expression of ER target genes, including SDF-1 itself. These effects were blocked by the antiestrogen ICI-182780 and by CXCR4 silencing, and conversely, estrogen-induced gene expression and growth of MCF-7 cells were impaired upon CXCR4 inhibition. Growth factor signaling also plays an important role in breast cancer. Overexpression and deregulated signaling of receptor tyrosine kinase ErbB2 correlate with aggressive breast tumor phenotype and poor outcomes. However, how ErbB2 and CXCR4 signaling is functionally related to regulate ER response in breast cancer cells is not known. Here we show that steady-state levels of ErbB2 and its dimeric partner ErbB3, as well as ErbB2 tyrosine phosphorylation were negatively regulated with the expression of CXCR4. CXCR4 downregulated ErbB2/ErbB3 dimer activation of the PI3-K/Akt pathway in response to ErbB3 ligand heregulin-β, whereas addition of SDF-1 restored activation levels. We found that heregulin-β promoted CXCR4 phosphorylation at serine 339, an important site for CXCR4 internalization and signaling. In addition, ErbB2 recruitment to CXCR4 was enhanced by ErbB3 and heregulin-β. Transcriptional activity and gene expression measurement showed that the hormonal repression of ER was relieved with the expression of CXCR4 and partially recuperated with the addition of SDF-1. Together, these results show that CXCR4 recruitment to ErbB2 alters ErbB2/ErbB3 signaling pathway and downstream regulation of ER hormonal activity in in breast cancer cells. Our work has enabled us to identify and characterize the impact of membrane receptors signaling on ER transcriptionnal response in breast cancer cells. Membrane signaling is one of the factors involved in endocrine therapy resistance and targeting the receptors implicated could be benificial to improve existing treatments and to work on the creation of new ones.

Chez la souris, la thérapie anti-HER2 est dépendante de la présence de cellules T CD8+IFN-γ+ et des réponses IFN de type I. Ces IFN sont induits par les TLRs suite à la reconnaissance de signaux de danger, appelés PAMPs et DAMPs. Les TLR-3 et TLR-9 sont tous deux de bons inducteurs d’IFN de type I et sont également capable d’agir en synergie afin d’augmenter les niveaux d’IFN-γ, de TNF-α et d’IL-12. Notre hypothèse fut que la stimulation de ces deux TLRs mènerait à l’amélioration de l’activité anti-tumorale du trastuzumab via le recrutement et l’activation des cellules immunitaires. Nos buts furent de confirmer le potentiel thérapeutique de la combinaison de l’anticorps anti-HER2, de l’agoniste de TLR-3, le poly(I:C), et de l’agoniste de TLR-9, le CpG ODN. Des études in vivo et in vitro nous ont permis de découvrir une synergie entre ces agents qui résulte en une cytotoxicité ciblée plus efficace. De plus, cette thérapie s’avéra efficace chez des modèles CD8-dépendants et CD8-indépendents. Les souris purent rejeter leur tumeur et demeurer sains plusieurs semaines après l’arrêt des injections. Ces souris étaient également protégées lors d’un challenge, soulignant ainsi la présence d’une immunité mémoire. Nous avons aussi découvert que l’administration combine de trastuzumab des deux agonistes de TLRs mène à des réponses systémiques. Des études de déplétion confirmèrent que les cellules T CD8+ sont cruciales pour la protection à long terme des animaux, mais que les pDC sont moins impliquées que ce que l’on pourrait croire. Leur absence n’a que modestement affecté les effets de notre thérapie. À l’opposé, les cellules NK sont d’importants médiateurs des effets thérapeutiques. Des expériences d’ADCC ont révélé que le CpG ODN et poly(I:C) ont tous deux la capacité d’améliorer les fonctions des cellules NK, mais que la stimulation simultanée des TLR-3 et TLR-9 permet de maximiser les effets bénéfiques du trastuzumab. De la même manière, l’addition de CpG ODN et de poly(I:C) aux anticorps anti-HER2 a permis d’augmenter les réponses pro-inflammatoires, plus spécifiquement l’IFN-γ, le TNF-α, l’IP-10 et l’IL-12. / In murine models, anti-HER2 therapy has been shown to be dependent on IFN-γ-producing CD8+ T cells and type I IFN responses. These IFN are induced by TLRs following the recognition of danger signals, called PAMPs or DAMPs. Both TLR-3 and TLR-9 are well known inducers of type I IFN and were also shown to act synergistically to enhance the levels of IFN-γ, TNF-α, and IL-12. Our hypothesis thus was that the stimulation of those two TLRs would lead to an enhancement of the activity of trastuzumab against tumor cells by the recruitment and activation of immune cells. Our goals were to assess the potential therapeutic effects of a combination of anti-HER2 mAbs, TLR-3 agonist poly(I:C) and TLR-9 agonist CpG ODN. In vivo and in vitro studies enabled us to discover a synergy between all agents resulting in a more efficient targeted cytotoxicity. Moreover, this therapy was fully effective in a CD8-dependant cell line as well as a CD8-independent one. Mice were able to completely reject their tumor and remain tumor-free weeks after the injections. Those mice were also protected against a rechallenge, thus underlining the presence of an immune memory. We also discovered that the combined administration of trastuzumab and the TLR agonists leads to systemic responses. Depleting studies confirmed that T CD8+ cells are crucial for the animal to remain tumor-free on the long term, but that pDC are far less involved than what we could have thought. Their absence only modestly affected the outcome of our therapy. On the counterpart, NK cells were important mediators of the therapeutic effect. ADCC assays revealed that both CpG ODN and poly(I:C) are able to enhance the functions of NK cells, but that simultaneous stimulation of the TLR-3 and TLR-9 allows to maximize the beneficial effects of trastuzumab. The same way, the addition of both CpG ODN and poly(I:C) to the anti-HER2 mAbs further enhanced pro-inflammatory responses, more specifically IFN-γ, TNF-α, IP-10 and IL-12.

The membrane mucin Muc4 is a heterodimer, bi-functional glycoprotein complex that is normally expressed in epithelial tissue. Functional studies on the extracellular mucin subunit of Muc4 have shown that it acts to promote anti-adhesion properties by sterically interfering with cell-cell and cell-matrix interactions and that the extent of this effect is directly associated with the number of tandem repeats on this subunit. Functional studies on the transmembrane subunit of Muc4 have shown that this subunit participates in intracellular signaling through interaction with the receptor tyrosine kinase ErbB2. This role of Muc4 was shown to be mediated by stabilizing the heregulin ligand-induced ErbB2-ErbB3 heterodimer through interference with the internalization process of these receptors, thus potentiating the PI3K, a survival-signaling pathway that is mediated by this heterodimer. However, Muc4 was also shown to potentiate ErbB2 phosphorylation in the absence of heregulin by an unknown mechanism. The aim of this work was to examine the role of Muc4 in intracellular signaling by evaluating the ligand-independent Muc4-ErbB2 interaction. Biochemical analyses of A375 human melanoma cells expressing Muc4 under different cell treatments, and probed with phospho-specific antibodies, were used to understand the mechanism. An antibody microarray screen was used to decipher the intracellular activated signaling pathways. The results of the mechanistic analysis indicated that Muc4 potentiates ErbB2 signaling significantly by interacting with ErbB2 and ErbB3 and by stabilizing the kinase active ErbB2 receptor, thus increasing its phosphorylation signal half-life and resulting in sustained ErbB2 signaling. The signaling pathway analysis suggests that through Muc4 direct interaction with ErbB2, signaling pathways that promote loss of cell polarity are activated. Loss of cell-cell adhesion is mediated by interference with the cadherin-catenin complex stability, and loss of cell-matrix adhesion is mediated by facilitating focal adhesion turnover. Together, these results suggest that Muc4 is a potent oncogenic factor, and further enhance our understanding of the role that Muc4 plays in ligand-independent intracellular signaling.

La régulation de la transcription des gènes par les récepteurs des estrogènes ERα et ERβ joue un rôle important dans la croissance cellulaire et dans le développement du cancer du sein. Une augmentation de l’expression de CXCR4 et de son ligand SDF-1/CXCL12 corrèle avec un phénotype plus agressif du cancer du sein. Ici, nous démontrons un mécanisme de boucle de régulation positive entre la signalisation de CXCR4/SDF-1 et l’activité transcriptionnelle des ERs dans des cellules cancéreuses mammaires. L’activité transcriptionnelle de ER et l’expression de gènes cibles de ER, dont SDF-1 lui-même, sont augmentées dans la lignée cancéreuse mammaire MCF-7 en réponse à SDF-1. Ces effets sont bloqués par l’anti-estrogène fulvestrant et par la délétion de CXCR4. Par ailleurs, l’expression des gènes et la prolifération des cellules cancéreuses mammaires MCF-7 en réponse à l’estrogène sont altérées par l’inhibition de CXCR4. La signalisation par les facteurs de croissance joue un rôle important dans le cancer du sein. La surexpression et la dérégulation de la signalisation par le récepteur à activité tyrosine kinase ErbB2 corrèlent avec un phénotype tumoral mammaire plus agressif et un moins bon pronostic. Cependant, comment la signalisation de ErbB2 et de CXCR4 sont fonctionnellement reliées dans la régulation de la réponse de ER dans les cellules cancéreuses mammaires n’est pas connue. Nous démontrons ici que CXCR4 régule négativement l’expression protéique de ErbB2 et de son partenaire d’interaction ErbB3 ainsi que la phosphorylation de ErbB2. CXCR4 altère l’activation de la voie PI3-K/Akt par le dimère ErbB2/ErbB3 en réponse à héréguline alors qu’en présence de SDF-1, les niveaux d’activation sont récupérés. Nous avons trouvé que héréguline-β promouvoit la phosphorylation de la sérine 339 de CXCR4, un site important pour l’internalisation et la signalisation du récepteur. De plus, le recrutement de ErbB2 à CXCR4 est favorisé par ErbB3 et héréguline-β. L’activité transcriptionnelle ainsi que l’expression des gènes cibles de ER en réponse à l’héréguline sont relevées avec l’expression de CXCR4 et partiellement récupérées avec l’addition de SDF-1. Ces résultats démontrent que le recrutement de CXCR4 à ErbB2 altère la signalisation médiée par ErbB2/ErbB3 ainsi que l’activité hormonale de ER dans des cellules cancéreuses mammaires. Nous travaux ont permis d’identifier et de caractériser l’impact de la signalisation médiée par des récepteurs membranaires sur la réponse transcriptionnelle de ER dans des cellules cancéreuses mammaires. La signalisation membranaire est un facteur pouvant contribuer à la résistance aux thérapies endocriniennes et donc cibler les récepteurs impliqués s’avèrerait utile pour améliorer les traitements existants et mettre au point de nouvelles approches. / Induction of estrogen-regulated gene transcription by estrogen receptors ERα and ERβ plays an important role in breast cancer development and growth. High expression of the chemokine receptor CXCR4 and its ligand CXCL12/SDF-1 has also been correlated with aggressive breast tumor phenotypes. Here, we describe a positive regulatory loop between CXCR4/SDF-1 signaling pathway and ER transcriptional competence in human breast cancer cells. Treatment of breast carcinoma MCF-7 cells with SDF-1 increased ER transcriptional activity and expression of ER target genes, including SDF-1 itself. These effects were blocked by the antiestrogen ICI-182780 and by CXCR4 silencing, and conversely, estrogen-induced gene expression and growth of MCF-7 cells were impaired upon CXCR4 inhibition. Growth factor signaling also plays an important role in breast cancer. Overexpression and deregulated signaling of receptor tyrosine kinase ErbB2 correlate with aggressive breast tumor phenotype and poor outcomes. However, how ErbB2 and CXCR4 signaling is functionally related to regulate ER response in breast cancer cells is not known. Here we show that steady-state levels of ErbB2 and its dimeric partner ErbB3, as well as ErbB2 tyrosine phosphorylation were negatively regulated with the expression of CXCR4. CXCR4 downregulated ErbB2/ErbB3 dimer activation of the PI3-K/Akt pathway in response to ErbB3 ligand heregulin-β, whereas addition of SDF-1 restored activation levels. We found that heregulin-β promoted CXCR4 phosphorylation at serine 339, an important site for CXCR4 internalization and signaling. In addition, ErbB2 recruitment to CXCR4 was enhanced by ErbB3 and heregulin-β. Transcriptional activity and gene expression measurement showed that the hormonal repression of ER was relieved with the expression of CXCR4 and partially recuperated with the addition of SDF-1. Together, these results show that CXCR4 recruitment to ErbB2 alters ErbB2/ErbB3 signaling pathway and downstream regulation of ER hormonal activity in in breast cancer cells. Our work has enabled us to identify and characterize the impact of membrane receptors signaling on ER transcriptionnal response in breast cancer cells. Membrane signaling is one of the factors involved in endocrine therapy resistance and targeting the receptors implicated could be benificial to improve existing treatments and to work on the creation of new ones.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 5 uppsatser. Även tryckt utgåva.

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