INTRODUÇÃO: o câncer de mama é uma doença heterogênea. Há necessidade de critérios diagnósticos e prognósticos mais refinados. O emprego da imunohistoquímica, através do painel prognóstico, fez despontar a figura do carcinoma triplonegativo e, da mesma forma, a histogenética trouxe à evidência o carcinoma basal. Paralelamente, o conhecimento sobre a origem biológica das neoplasias e da sua heterogeneidade vem sendo acentuadamente debatido através do tema das células-tronco tumorais. OBJETIVOS: investigar a prevalência de carcinomas basais e pentanegativos, numa amostra de carcinomas triplonegativos, e estabelecer associações com a expressão de células-tronco tumorais nestes tumores. Verificar diferenças entre estes subtipos com as variáveis clinicopatológicas. MÉTODOS: 94 carcinomas mamários triplonegativos foram testados para CK5/6, HER1, CD44 e CD24. As expressões desses marcadores por imuno-histoquímica automatizada foram avaliadas através de escore simples de positividade (porcentagem de células) e correlacionadas com os dados clínico-patológicos e análise de sobrevivência. RESULTADOS: carcinomas basais apresentam maior grau tumoral que carcinomas pentanegativos (p=0,004). A negatividade para CD44 (p=0,007) e o perfil CD44- CD24+ (p=0,013) foram associados com maior invasão vascular entre carcinomas triplonegativos. A expressão de CD44 foi associada aos carcinomas basais (p=0,007). O perfil CD44-CD24-/low foi associado aos carcinomas pentanegativos (p=0,04). Nenhuma das variáveis em estudo foi associada com os desfechos clínicos. CONCLUSÃO: Carcinomas mamários basais e pentanegativos são subtipos tumorais bastante próximos. Nosso estudo é o primeiro desenhado especificamente para avaliar a presença células tronco-tumorais mamárias entre carcinomas basais e pentanegativos, onde o perfil CD44-CD24-/low foi associado ao subtipo pentanegativo, e o perfil CD44-CD24+ à invasão vascular, resultados que merecem confirmação por histogenética em estudos de maior porte. / INTRODUCTION: Breast cancer is a heterogeneous disease, and there is a need for more refined diagnostic and prognostic criteria. Immunohistochemistry, as a breast prognostic panel, has given rise to triple-negative carcinoma, as well as histogenetics highlighted basal carcinoma. Concomitantly, the understanding of the biological origins of neoplasia and its heterogeneity has been strongly debated through the theme of cancer stem cells. OBJECTIVES: To investigate the prevalence of basal and penta-negative carcinomas in a sample of triple-negative carcinomas and to establish associations with cancer stem cells (CD44/CD24 expression profiles) and the clinicopathological variables within these tumors. METHODS: Ninety-four triplenegative breast carcinomas were tested for CK5/6, HER1, CD44 and CD24. The expression of these markers was evaluated by automated immunohistochemistry using a simple positivity score (percentage of cells) and was correlated with the clinicopathological and survival analysis data. RESULTS: Basal carcinomas had higher tumor grades than penta-negative carcinomas (p=0.004). CD44 negativity (p=0.007) and the CD44-CD24+ phenotype (p=0.013) were associated with increased vascular invasion amongst the triple-negative carcinomas. CD44 expression was associated with basal carcinomas (p=0.007). The CD44-CD24-/low phenotype was associated with penta-negative carcinomas. None of the variables in the study were associated with clinical outcome. CONCLUSION: Basal and penta-negative breast carcinomas are closely related tumor subtypes. Our study is the first to be specifically designed to assess the presence of breast cancer stem cells in basal and pentanegative carcinomas. The CD44-CD24-/low phenotype was associated with the pentanegative subtype, and the CD44-CD24+ phenotype was associated with vascular invasion. These results require histogenetic confirmation in larger studies.

The chondroitin sulphate proteoglycan (CSPG) versican forms complexes with hyaluronan (HA), which are essential in a range of functions including cellular proliferation and migration. Four isoforms of versican result from alternative splicing. Furthermore, biological roles have been identified for the proteolytic cleavage product of versican which contains the N-terminal G1 hyaluronan binding domain. All of these versican forms have different tightly regulated tissue expression profiles. Consequently, impaired regulation is associated with a number of disease pathologies. For example the largest variants (V0/V1) have been shown to be negative indicators of disease outcome in a number of malignant cancers and are a marker of disease progression in atherosclerosis. Interestingly, the smaller versican isoform V3 which lacks CS chains has been demonstrated to have the potential to reverse disease associated phenotypes. The motivation for carrying out the work in this thesis was to try and gain a better understanding of how versican functions on a molecular scale. In this regard, the first aim was to investigate the structure of the hyaluronan binding region of versican using a construct called VG1. The structure of VG1 was analysed in the presence and absence of hyaluronan oligomers. This revealed an insight into the multi-modular structure of the versican hyaluronan binding region and demonstrated that on binding to HA, VG1 under goes a conformational change. Furthermore, the interaction between VG1 and longer lengths of hyaluronan (pHA) was investigated. This demonstrated that when VG1 binds to pHA it is does so with positive cooperativity, packing very close to neighbouring VG1 molecules along a chain of HA. One consequence of this interaction was to reorganise pHA into a helical conformation, an organisation that was confirmed by a number of solution phase techniques. The effect of this reorganisation of pHA by VG1 on HA/CD44 interactions was also assessed. Previously the interaction between CD44 (a cell surface hyaluronan receptor) and long chains of HA (>30 kDa) was shown to be irreversible; however we demonstrate that VG1 can reverse this. Furthermore, a TSG-6 enhanced CD44/interaction was also completely reversed by the addition of VG1. This provides an indication that a functional hierarchy of hyaluronan binding proteins may exist which could have important implications in understanding the function of hyaluronan complexes. Currently, we do not know whether intact versican molecules could interact with HA in the same way as VG1. However, preliminary data suggests that the CS-containing variants (i.e. V0, V1 and V2) would not, whereas V3 and versican fragments could. This work provides an exciting mechanistic insight into the function of versican variants and their breakdown products.

This project focused on the design of a cellular scaffold applicable for the promotion of peripheral nerve regeneration. Firstly, we established a correlation between the organization of HA/PEG co-polymeric networks to their mechanical and degradability properties; cell adhesion was conferred to all gels by the incorporation of RGD peptides. Three families of hydrogels were produced using different procedures to permit an increasing physical incorporation of HA into a PEGDA-based network. From a comparative study of rheological properties and enzymatic degradability, co-networks obtained using thiolated HA as chain transfer agent during PEGDA photo-polymerization were selected for further biological investigations, aiming to link the cellular response of L929 murine fibroblasts (phenotype, proliferation rate, metabolic activity) to the composition and the consistency of selected hydrogels. Our findings showed that there is a clear relation between increasing hardness and increasing cell spreading/proliferation rate. This study illustrated the possibility to fine tune cell/material interactions with appropriate reactive processing techniques. As a spin-off of this study, we become interested in the interplay of cellular interactions in the use of materials that contain both HA and RGD peptides, which can bind at the same time to HA receptors such as CD44 and av integrins. We focused on soluble HA derivatives, with or without dandling RGD peptides. The kinetics and the mechanistic details of both HA and HA-RGD internalization were studied in a phagocytic model (J774.2 murine macrophages). HA-RGD showed a form of synergic binding to integrins and CD44 (HA receptor), whereas its uptake remained solely regulated by CD44 dwell-time on the cell membrane. This study demonstrated that the knowledge of the rate-determining steps of the uptake of a carrier is necessary for assessing its efficiency. In this case, the presence of multiple ligands on a carrier was beneficial in some respect, but may not be optimal to overcome internalization limitations that arise from the slow turnover of the determining receptor. Finally, we studied the relation between the regulation of the expression of CD44 / RHAMM (HA receptor mediated motility) and the motility of Schwann cells (peripheral glial cells) and stem cells differentiated into a glial phenotype. Rt-PCR and immuno-assay experiments suggested that RHAMM up-regulation is associated with glial differentiation and we speculate that in the future this HA receptor could be considered as a differentiation marker. We also illustrated the importance of HA / RHAMM interaction for the motility of glial cells. These results indicate the importance of HA in mediating glial cell function during peripheral nerve regeneration and have implications for therapeutic repair strategies.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22812号 / 生博第446号 / 新制||生||59(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 原田 浩, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.

Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. It is hypothesized that the CD44-HA interaction regulates proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA. To address this hypothesis, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50 kDa (low molecular weight; LMW-HA) and 500-750 kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results emphasize the importance of presentation mode and molecular weight specificity in biomaterial studies on the impact of HA on cell behavior.

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with β1 or β3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Despite progress being made in our understanding of triple negative breast cancer (TNBC), the overall survival and disease-free survival for TNBC patients continues to be considerably poorer than their ER/PR/HER2+ counterparts. Metastasis and chemoresistance are the pivotal issues holding back the long-term success of TNBC treatments. In addition to the bulk tumor cells, cancer stem cells (CSCs) have emerged as important targets for alleviating TNBC progression and relapse. Cisplatin, a platinum based chemotherapeutic agent, has shown promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with tumor hypoxia that in turn promotes CSC enrichment and drug resistance. My work is to develop a combinational treatment to improve the long-term therapeutic potential of cisplatin that not only targeted the bulk TNBC population but also ALDHhigh and CD44+/CD24- CSC populations. Through clinical dataset analysis, I found that patient TNBC tumors expressed high levels of epidermal growth factor receptor (EGFR) and hypoxia genes. A similar expression pattern was demonstrated in cisplatin-resistant ovarian cancer. I therefore developed a combinational therapeutic to co-inhibit EGFR and hypoxia using metformin (an AMPK activator) and gefitinib (an EGFR inhibitor), which sensitized bulk TNBC cells to cisplatin and also led to the effective inhibition of both CD44+/CD24- and ALDHhigh CSCs. I obtained similar results by using clinically relevant TNBC patient samples ex vivo. Since these drugs are already frequently used in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to improve the long-term therapeutic outcome of cisplatin for TNBC treatment.

Intratumor heterogeneity poses a significant challenge for the diagnosis and treatment of patients with breast cancer because distinct sub-populations of tumor cells contribute significantly more to therapy resistance and tumor recurrence than others. Consequently, understanding the mechanisms that contribute to this heterogeneity and identifying sub-populations responsible for aggressive behavior is a significant and timely problem. Considerable evidence indicates that a subpopulation of tumor cells with stem/progenitor-like characteristics, termed cancer stem cells (CSCs), is responsible for therapy resistance and recurrence, sparking interest in characterizing novel biomarkers and therapeutic targets for this aggressive population of cells. Unfortunately, CSCs share many protein markers with normal mammary stem/progenitor populations, minimizing potential targets for diagnostic and therapeutic purposes. Therefore, in my thesis research, I investigated novel ways to identify CSC populations based on their glycome. I observed that breast CSCs have a unique glycosylation pattern that can be used to distinguish them from other tumor populations. Specifically, I discovered a novel α2,3 sialoglycan on Core2 O-linked glycans expressed on CSCs that can identified using the lectin SLBR-N. I found that SLBR-N can be used to distinguish CSCs from bulk tumor cells in multiple in vitro and in vivo models. I also discovered that the CSC marker, CD44s, expresses O-linked α2,3 sialoglycan and that this glycan alters CD44s function by promoting the activation of the PDGFRβ/STAT3 pathway. In contrast, the fucosyltransferase FUT3 and its glycan sialyl Lewis X (sLeX) are expressed on non-CSCs and they function to impede stemness by inhibiting CD44s-mediated PDGFRβ/STAT3 signaling. In summary, this thesis provides insights into glycan heterogeneity in breast cancer and novel ways to identify CSCs using the glycome.

Structured abstract Hypothesis Cancer stem cells (CSCs) are subpopulations of cells which could contribute to tumor growth, metastasis formation and chemoresistance. CSCs can be detected by surface markers assessed by immunohistochemistry methods. A typical surface marker for CSCs is CD44 (standard form). We assumed, that CD44(s) could serve as a prognostic factor and marker of chemoresistance in patients with epithelial ovarian cancer. The aim of study 1. To recruit group of patients with histologically verified epithelial ovarian carcinoma. 2. To evaluate prognostic significance of known prognostic factors in our series of patients. 3. To assess the expression of CD44 in specimens of primary tumors and specimens of implantation metastasis using immnunohistochemistry and analyze their correlation. 4. To evaluate the expression of CD44 in relation to known prognostic factors. To analyze the significance of CD44 expression evaluation for overall survival, disease-free interval and chemoresistance. To find CD44 positivity cut-off by using statistical methods Materials and Methods A retrospective study was performed on 87 patients with histologically verified EOC. All patients were tested for primary tumor specimens, 48 of them were tested with regard to both specimens of primary tumor and implantation...