名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程） 学位授与年月日:平成20年3月25日

Le traitement du mélanome métastatique a été révolutionné par le développement de thérapies ciblées, qui ont montré un bénéfice significatif sur la survie globale. En particulier, l'inhibition de la sérine-thréonine kinase BRAF, mutée dans 60% des mélanomes, par le Vémurafénib (PLX4032), a montré un gain de survie de 6 à 8 mois comparée à la chimiothérapie de référence, la Dacarbazine. Cependant, une très faible proportion de patients répond sur le long terme. En effet, la majorité des patients développent un échappement thérapeutique dans un délai médian de 6 mois. Des mécanismes cellulaires ont été mis en évidence dans l'apparition de cette résistance acquise, notamment l'implication de MITF, un facteur de transcription majeur des mélanocytes, ainsi que des modifications de l'expression de plusieurs membres de la famille de Bcl-2. Cependant, une meilleure compréhension des mécanismes de résistance aux thérapies anti-BRAF semble essentielle, tout comme l'utilisation de nouvelles approches thérapeutiques combinées afin d'optimiser l'efficacité des traitements et la durée du bénéfice clinique. Notre groupe a récemment identifié des altérations du métabolisme du céramide et de l'un de ses dérivés, la Sphingosine 1-phosphate (S1P), dans les cellules de mélanome humain comparé à des mélanocytes sains. En effet, nous avons montré que la S1P lyase (SPL), qui dégrade irréversiblement la S1P est sous exprimée. Au contraire, l'expression de la sphingosine kinase 1 (SK1), qui produit la S1P, est augmentée dans les cellules de mélanome, conséquence directe de la mutation BRAF. Ces perturbations ont pour effet d'augmenter les niveaux de S1P. Ce lysophospholipide favorise la survie cellulaire ainsi que la résistance vis-à-vis d'agents thérapeutiques dans diverses cellules tumorales. L'objectif de cette thèse a été d'évaluer si le métabolisme de la S1P peut moduler la résistance acquise des cellules de mélanome humain aux inhibiteurs de BRAF. Nous avons montré que la surexpression de la SPL ou l'inhibition pharmacologique de la SK1 (SKI-I) sensibilise les mélanomes métastatiques à l'apoptose induite par la thérapie ciblée. Ce phénomène est associé à une diminution de MITF et de l'une de ses cibles directes, la protéine anti-apoptotique Bcl-2. La diminution d'expression protéique de MITF peut être réversée par un traitement de S1P exogène. De plus, nous avons montré pour la première fois une augmentation de l'expression des récepteurs 1 et 3 à la S1P (S1PR1 et S1PR3), dans les cellules de mélanome présentant une résistance acquise au PLX4032. Ces modifications sont associées à l'expression accrue de MITF. La surexpression de la SPL, le traitement par le SKI-I ou par des inhibiteurs ciblant les S1PR1 et S1PR3, surmonte la résistance acquise de ces cellules au PLX4032 via la diminution d'expression des S1PRs, de MITF, et de Bcl-2. Par conséquent, en contrôlant l'expression de protéines clés de la survie et de la résistance, le métabolisme de la S1P représente une nouvelle approche thérapeutique pour améliorer l'efficacité des thérapies ciblées. / The treatment of metastatic melanoma has changed considerably in recent years with the development of targeted therapies, which have shown a significant benefit in overall survival. In particular, the inhibition of the frequently mutated serine-threonine kinase BRAF, by Vemurafenib (PLX4032) showed that survival rates increase by 6 to 8 months compared to standard chemotherapy, Dacarbazine. However, a very small proportion of patients will respond to the long term, and the majority of patients relapses in a median of 6 months. Cellular mechanisms have been identified in the appearance of this acquired resistance, including the involvement of MITF, a major transcription factor of melanocytes, as well as changes in the expression of several members of Bcl-2 family. However, a better understanding of these mechanisms seems essential, as is the use of new therapeutic strategies to optimize treatment efficacy and duration of clinical benefit. Our group recently showed some alterations of ceramide metabolism and its derivative sphingosine 1-phosphate (S1P) in human melanoma cells compared to healthy melanocytes. For instance, S1P lyase (SPL), which degrades S1P, is under-expressed. Conversely, sphingosine kinase 1 (SK1), which produces S1P, is over-expressed in tumor cells, as a direct result of BRAF mutation. These alterations increases the levels of S1P. This lysophospholipid promotes cell survival and the resistance to therapeutic agents in a variety of tumor cells. This PhD project aimed at defining whether S1P metabolism could modulate the resistance of human melanoma cells to PLX4032. Here, we show that SPL overexpression or pharmacological inhibition of SK1 by SKI-I sensitizes metastatic melanoma cells to PLX4032-induced apoptosis. This phenomenon is associated with a decreased expression of the master regulator of melanocyte differentiation MITF as well as its direct cellular target Bcl-2. The decrease in MITF protein can be reversed by treating cells with exogenous S1P. Interestingly, we also report for the first time an increased expression of SK1 as well as the S1P receptors, S1PR1 and S1PR3, in melanoma cells with acquired resistance to PLX4032 as compared to sensitive counterparts. These modifications are associated with high expression of MITF. Overexpression of SPL, treatment with SKI-I or antagonists of S1PR1 ans S1PR3, strongly overcomes acquired resistance to PLX4032 through a decrease in the expression of S1PR, MITF as well as Bcl-2. Thus, by controlling the expression of key proteins in melanoma cell survival and resistance, S1P metabolism could represent a new therapeutic approach to enhance the effectiveness of targeted therapies.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to define biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical markers in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, the results showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs. / Master of Science

Computationally driven drug design/discovery efforts generally rely on accurate assessment of the forces that guide the molecular recognition process. HINT (Hydropathic INTeraction) is a natural force field, derived from experimentally determined partition coefficients that quantifies all non-bonded interactions in the biological environment, including hydrogen bonding, electrostatic and hydrophobic interactions, and the energy of desolvation. The overall goal of this work is to apply the HINT-based atomic level description of molecular systems to biologically important proteins, to better understand their biochemistry – a key step in exploiting them for therapeutic purposes. This dissertation discusses the results of three diverse projects: i) structural modeling of human sphingosine kinase 2 (SphK2, a novel anticancer target) and binding mode determination of an isoform selective thiazolidine-2,4-dione (TZD) analog; ii) structural modeling of human cytomegalorvirus (HCMV) alkaline nuclease (AN) UL98 (a novel antiviral target) and subsequent virtual screening of its active site; and iii) explicit treatment of interfacial waters during protein-protein docking process using HINT-based computational tools. SphK2 is a key regulator of the sphingosine-rheostat, and its upregulation /overexpression has been associated with cancer development. We report structural modeling studies of a novel TZD-analog that selectively inhibits SphK2, in a HINT analysis that identifies the key structural features of ligand and protein binding site responsible for isoform selectivity. The second aim was to build a three-dimensional structure of a novel HCMV target – AN UL98, to identify its catalytically important residues. HINT analysis of the interaction of 5’ DNA end at its active site is reported. A parallel aim to perform in silico screening with a site-based pharmacophore model, identified several novel hits with potentially desirable chemical features for interaction with UL98 AN. The majority of current protein-protein docking algorithms fail to account for water molecules involved in bridging interactions between partners, mediating and stabilizing their association. HINT is capable of reproducing the physical and chemical properties of such waters, while accounting for their energetic stabilizing contributions. We have designed a solvated protein-protein docking protocol that explicitly models the Relevant bridging waters, and demonstrate that more accurate results are obtained when water is not ignored.

Cholangiocarcinoma (CCA) has a high mortality rate and its occurrence is rising. This increase prompts the need for improved CCA treatments. Studies have suggested that CCA is highly reliant on the sphingosine-1-phosphate-receptor-2 (S1PR2) and sphingosine kinase 2 (SphK2). Recently, a competitive SphK2 inhibitor, ABC294640, has been approved for clinical trial. ABC294640 has the potential to treat CCA, which is support by a phase I clinical study that was able to temporarily treat a patient suffering from metastasized CCA with ABC294640. To determine the viability of ABC294640 as a treatment for CCA, this study focused on determining the effects of ABC294640 on rat CCA cell lines. We found that ABC294640 inhibited the growth and migration of CCA and CAFs cells. The growth and count of 3-D organotypic co-culture of CCA and CAFs, which forms the “duct-like” structures, were reduced by ABC294640. The potential of inhibiting SphK2 as a treatment for CCA is supported by our finding of increased expression of S1PR2 and SphK2 in CCA patient liver samples. In conclusion, ABC294640 represents a potential therapeutic agent for CCA.