京都大学 / 新制・課程博士 / 博士(医学) / 甲第25194号 / 医博第5080号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 小濱 和貴, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24884号 / 医博第5018号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 実, 教授 小濱 和貴, 教授 中島 貴子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Pancreatic cancer is the fourth leading cause of cancer-related deaths in the USA. Asymptomatic early cancer stages and late diagnosis leads to very low survival rates of pancreatic cancers, compared to other cancers. Treatment options for advanced pancreatic cancer are limited to chemotherapy and/or radiation therapy, as surgical removal of the cancerous tissue becomes impossible at later stages. Therefore, there's a critical need for innovative and improved chemotherapeutic treatment of (late) pancreatic cancers. It is mandatory for successful treatment strategies to overcome the drug resistance associated with pancreatic cancers. Nanotechnology based drug formulations have been providing promising alternatives in cancer treatment due to their selective targeting and accumulation in tumor vasculature, which can be used for efficient delivery of chemotherapeutic agents to tumors and metastases. The research of my thesis is following the principle approach to high therapeutic efficacy that has been first described by Dr. Helmut Ringsdorf in 1975. However, I have extended the use of the Ringsdorf model from polymeric to nanoparticle-based drug carriers by exploring an iron / iron oxide nanoparticle based drug delivery system. A series of drug delivery systems have been synthesized by varying the total numbers and the ratio of the tumor homing peptide sequence CGKRK and the chemotherapeutic drug doxorubicin at the surfaces of Fe/Fe₃O₄-nanoparticles. The cytotoxicity of these nanoformulations was tested against murine pancreatic cancer cell lines (Pan02) to assess their therapeutic capabilities for effective treatments of pancreatic cancers. Healthy mouse fibroblast cells (STO) were also tested for comparison, because an effective chemotherapeutic drug has to be selective towards cancer cells. Optimal Experimental Design methodology was applied to identify the nanoformulation with the highest therapeutic activity. A statistical analysis method known as response surface methodology was carried out to evaluate the in-vitro cytotoxicity data, and to determine whether the chosen experimental parameters truly express the optimized conditions of the nanoparticle based drug delivery system. The overall goal was to optimize the therapeutic efficacy in nanoparticle-based pancreatic cancer treatment. Based on the statistical data, the most effective iron/iron oxide nanoparticle-based drug delivery system has been identified. Its Fe/Fe₃O₄ core has a diameter of 20 nm. The surface of this nanoparticle is loaded with the homing sequence CGKRK (139-142 peptide molecules per nanoparticle surface) and the chemotherapeutic agent doxorubicin (156-159 molecules per surface), This nanoplatform is a promising candidate for the nanoparticle-based chemotherapy of pancreatic cancer.

The development of several organ systems through modeling and shaping of the tissue structure occurs from signaling through axon guidance molecules. The Slit family of ligands has been shown to regulate branching morphogenesis in mammary gland duct development and loss of Slit gene expression during this time leads to the formation of hyperplastic, disorganized lesions suggesting a potential role for Slits in cancer formation. Characterization of human pancreatic ductal adenocarcinoma cell lines showed a loss of SLIT2 expression in cells that contain activated Kras. Loss of SLIT2 expression was associated with DNA methylation of CpG sites within the SLIT2 core promoter and chromatin enrichment of repressive histone modifications at the SLIT2 transcriptional start site. Additionally, treatment of pancreatic ductal adenocarcinoma cell lines with demethylating agent 5-aza-2'-deoxycytidine led to SLIT2 re-expression while treatment with histone deacetylase inhibitor Trichostatin A did not. Mir-218-1 is an intronic microRNA encoded within intron 15 of the SLIT2 gene. Expression of mir-218-1 does not correlate with SLIT2 mRNA expression suggesting that it is transcribed from a promoter independent of the SLIT2 gene promoter. Pancreatic ductal adenocarcinoma cell lines showed a peak of H3K4me3 chromatin enrichment localized to a 1kb region within intron 4 of the SLIT2 gene denoting a candidate alternative promoter for mir-218-1. A concordant peak of H4ac chromatin enrichment overlapped the peak of H3K4me3 enrichment and transcriptional activity was measured from the 1kb region in all pancreatic ductal adenocarcinoma cell lines. A NF-κB binding site was also predicted to exist within the 1kb region. Transfection with two independent siRNAs to NF-κB led to an increase in both pre-mir-218-1 and mature mir-218-1 while treatment with an inhibitor to IκB kinase led to an increase in pre-mir-218-1 expression. Additionally, the p65 subunit of NF-κB was found to bind to the candidate mir-218-1 alternative promoter in pancreatic ductal adenocarcinoma cell lines that do not contain DNA CpG methylation at the predicted NF-κB binding site. It was discovered that miR-218 is a modulator of ARF6 expression suggesting a role in the inhibition of pancreatic ductal adenocarcinoma cell invasion through modulation of the actin cytoskeleton. Overexpression with a miR-218 precursor showed that miR-218 is an inhibitor of pancreatic ductal adenocarcinoma cell invasion in two dimensions. Additionally, it was found that while miR-218 does not have an affect on the ability of pancreatic ductal adenocarcinoma cells to form functional invadopodia, miR-218 is an inhibitor of the extracellular matrix degradation properties of mature invadopodia. Interestingly, the effect of miR-218 on pancreatic ductal adenocarcinoma cell invasion or extracellular matrix degradation is not reliant on the cell's dependency on Kras signaling for growth and survival. Collectively, these observations indicate that understanding the transcriptional regulation of SLIT2 and mir-218-1 expression as well as their signaling properties may provide a step toward the development of diagnostic tests and therapeutic treatments for patients with invasive or metastatic pancreatic ductal adenocarcinoma.

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs). There is accumulating evidence that PSCs influence the malignant phenotype of PDAC. The aim of this study was to analyse the tumour response to radiation treatment in the presence of PSCs and to investigate the cytokine network in the coculture of PSCs and pancreatic cancer cells (PCCs). PSCs were used in coculture with different PCC lines. Clonogenic survival assays of several PCC lines cocultured with PSCs showed decreased radiosensitivity. This effect was abrogated by inhibition of the β1-integrin/FAK signalling pathway. Furthermore, tumour regrowth experiments after irradiation showed that coinjected PSCs were radioprotective for PCCs after single-dose and fractionated irradiation in xenografts. In addition, we examined the expression of 50 proteins in the supernatants of PCCs and PSCs in mono- and coculture conditions. The detected cytokine expression profile of PSCs included many proinflammatory factors. Also, we identified IP-10 as the chemokine with the highest differential upregulation in PSCs by paracrine stimuli from five different PCC lines. Human PDAC with a high stroma component had elevated IP-10 mRNA expression. IP-10 did not stimulate tumour cell growth and migration in our conditions even though several PCCs expressed its cognate receptor CXCR3. Nevertheless, we discovered that in human PDAC samples IP-10 and CXCR3 mRNA levels correlated with the presence of CD3ε, CD4, FoxP3, CTLA4 and CD39 used as surrogate markers for T regulatory cells (Tregs), known to exert an immunosuppressive effect. In conclusion, these data demonstrate that PSCs enhance survival of PCCs to radiation by activating β1-integrin/FAK signalling. Furthermore, the interaction between the tumour stroma in pancreatic cancer may support an immunosuppression by chemoattraction of Tregs following upregulation of IP-10. Further characterisation of the paracrine signalling between PCCs, PSCs and immune cells will improve the understanding of pancreatic cancer biology and could lead to the identification of new targets for multimodal therapy.

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained. Additionally, the application of an ultrasound gel to the imaging surface may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate imaging of tissues and cells with high resolution and contrast. We have recently developed a novel method for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells using MPM. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.

Integrin α6β4 is upregulated in pancreatic carcinoma, where signaling promotes metastatic properties, in part by altering the transcriptome. Such alterations can be accomplished through DNA demethylation of specific promoters, as seen with the pro-metastatic gene S100A4. I found that signaling from integrin α6β4 dramatically upregulates expression of amphiregulin (AREG) and epiregulin (EREG), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if AREG and EREG are regulated by DNA methylation, pancreatic cancer cells with low AREG and EREG expression were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR), resulting in stable overexpression of AREG and EREG, and this induction required signaling from integrin α6β4. Similarly, treatment of cells with high integrin α6β4 with the methyl donor S-adenosylmethionine inhibited gene expression of AREG and EREG. Whole genome bisulfite sequencing on pancreatic cancer cells reveled hypomethylation of the promoter regions of AREG and EREG when integrin α6β4 is high, and these regions correspond to H3K27Ac, indicative of enhancer location. Interestingly, I also observed genome-wide DNA demethylation, and a large proportion of altered CpGs correspond to potential enhancers. It is currently accepted that active DNA demethylation occurs via DNA repair. I tested this hypothesis by treating cells with Gemcitabine, which inhibits multiple components of DNA repair, including DNA demethylation mediated by GADD45A. Gemcitabine treatment resulted in marked reduction in AREG and EREG expression. To further test the involvement of GADD45A, I used RNAi-mediated knockdown or cDNA overexpression to alter GADD45A levels. In both instances, AREG and EREG expression positively correlated with GADD45A, particularly when integrin α6β4 is high, indicating that GADD45A is a rate-limiting step in AREG and EREG overexpression. Similarly, using stable shRNA, I show that Thymine DNA Glycosylase (TDG), and TET1 known modulators of DNA demethylation, are required for AREG and EREG expression in integrin α6β4 high cells, and nuclear localization of TDG is much higher in cells with high integrin α6β4. Using a specific inhibitor I found that AREG and EREG expression is dependent on Parp-1. Finally, I determined that integrin α6β4 signaling enhances cells ability to respond to and survive in the presence of DNA damage, and that active DNA repair is required for integrin α6β4 mediated DNA demethylation. Taken together, these data indicate that DNA repair is required to maintain overexpression of AREG and EREG in response to signaling from integrin α6β4 and that integrin α6β4 promotes this overexpression by enhancing DNA repair.

Das Pankreaskarzinom ist eine der aggressivsten Krebsarten mit einer extrem schlechten Prognose für die Patienten (Robert Koch-Institut 2012). Der Entwicklung neuer Substanzen für die Therapie wird eine große Bedeutung zur Verbesserung der Behandlungsergebnisse zugesprochen (Li D et al. 2004). Das Ziel der vorliegenden Dissertation war, die Wirkung des für die Behandlung von Bandwurminfektionen eingesetzten Anthelmintikums Niclosamid auf verschiedene Pankreaskarzinom-Zelllinien zu untersuchen. Die humanen Pankreaskarzinom-Zelllinien (MIA PaCa-2, Panc-1 und L3.6pl) wurden mit aufsteigenden Konzentrationen des Medikaments Niclosamid für 24 Stunden be¬handelt (0,1; 1; 3; 10; 30 µM, Inkubation in RPMI-1640-Medium). Nachfolgend wurden die vitalen Zellen unter dem Lichtmikroskop ausgezählt. Mit Hilfe einer modifizierten Boyden-Kammer wurde die Migration der Zellen durch eine poröse Membran (Poren¬größe 8 µm) untersucht. FACS-Analysen nach Färbung mit Annexin und Propidium¬iodid dienten zur Bestimmung der Apoptose- und Nekroserate sowie zum Nachweis der Veränderungen im Zellzyklus. Um Effekte von Niclosamid auf die Signaltrans¬duktion aufzuzeigen, wurden qRT-PCR-Analysen ausgewählter Wnt- und Hedgehog-Zielgene durchgeführt. Bereits eine Konzentration von 3 µM Niclosamid führte zu einer signifikanten Reduktion der vitalen Zellen in allen drei Zelllinien. Bei MIA PaCa-2 und Panc-1 konnte sogar ab 0,1 µM Niclosamid eine signifikante Abnahme der Zellzahl beobachtet werden (MIA PaCa-2 auf 48 % und Panc-1 auf 55 %); p≤0,05. Außerdem konnte bei einer Konzentration von 0,5 µM eine Inhibition der Zellmigration in MIA PaCa-2 auf 28 % und Panc-1 auf 27 % des Ausgangswertes gezeigt werden. In der FACS-Analyse zeigte sich bei Konzentrationen von 1 µM (Panc-1) und 10 µM (MIA PaCa-2 und L3.6pl) Niclosamid eine signifikante Zunahme der apoptotischen und nekrotischen Populationsanteile. Weiterhin konnte eine dosisabhängige Induk-tion des Zellzyklusarrestes bei Konzentrationen zwischen 0,1-1 µM beobachtet wer-den. Signifikante Effekte auf die Expression der Wnt-Zielgene fanden sich bei allen drei Zelllinien. So kam es bei mit Niclosamid behandelten Zellen zu einem Rückgang der Expression der kanonischen Wnt-Komponenten Axin2 und MMP7 (Panc-1, L3.6pl), Cyclin D1 (MIA PaCa-2, Panc-1, L3.6pl) und BCL9 (MIA PaCa-2). Eine ein¬deutige Beeinflussung der Komponenten des nicht-kanonischen Wnt-Signalweges (c-jun, Wnt5a) und des Hedgehog-Signalweges (SHH, SMO, Ptch1, Gli1) konnte nicht ermittelt werden. Niclosamid hatte demnach mindestens drei verschiedene Wirkmechanismen: Es hemmte die Zellproliferation und Migration, förderte die Apoptose und Nekrose und be¬wirkte einen Zellzyklusarrest der Pankreaskarzinom-Zellen. Außerdem konnte mit der PCR der hemmende Einfluss von Niclosamid auf den kanonischen Wnt-Signalweg, einen der wichtigsten Signalwege in der Entwicklung des Pankreaskarzinoms, aufge¬zeigt werden. Zusammenfassend lässt sich sagen, dass in Niclosamid ein neuer In¬hibitor des Wnt-Signalweges in Pankreaskarzinom-Zelllinien identifiziert werden konnte. Die Ergebnisse der vorliegenden Dissertation berechtigen möglicherweise zur Annahme, dass Niclosamid zukünftig eine aussichtsreiche chemotherapeutische Substanz zur Behandlung des Pankreaskarzinoms darstellen könnte.