No / Improved understanding of the involvement of matrix metalloproteinases (MMPs), including membrane-type MMPs (MT-MMPs), in human tumours has potential diagnostic, prognostic and therapeutic implications. We assessed the relationship between MT-MMP expression and clinicopathological parameters in human non-small cell lung cancer (NSCLC) and histologically normal lung tissue by quantitative Real Time PCR (qRT-PCR). All MT-MMPs (MMPs 14-17, 24 and 25) were detected by qRT-PCR with significantly higher MMP-14, -15 and -17 expression observed in tumour relative to normal lung specimens. MMP-16 was undetectable in normal lung but expressed in 8% tumours. MMP-15 demonstrated significant overexpression in adenocarcinomas relative to squamous cell carcinomas and normal lung tissue. MMP-14 mRNA expression strongly correlated to MMP-14 proteolytic activity in preclinical tumour models, indicating that qRT-PCR may predict MMP-14 activity levels in NSCLC. These data suggest that MMP-14, -15 and -17 may be good markers of disease, or therapeutic targets for treatment of human NSCLC.

Yes / Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer. / Cancer Research UK Project Grant (C16929/A14402) and the Elizabeth Blackwell Institute, through its Wellcome Trust ISSF Award (105612/Z/14/Z). The Imperial NIHR/Biomedical Research Centre and the CR-UK/Dept of Health funded Imperial Experimental Cancer Medicine Centre.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第25230号 / 薬科博第192号 / 新制||薬科||21(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 樋口 ゆり子, 教授 二木 史朗 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25492号 / 医博第5092号 / 新制||医||1073(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 斎藤 通紀, 教授 浅野 雅秀, 教授 柊 卓志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of this thesis was to evaluate and compare the peripheral dose associated with VMAT lung SABR treatments for 10X, 6X, and 10X-FFF beams. Flattening Filter Free (FFF) radiotherapy photon beams exhibit high dose rates as compared to standard flattened photon beams. The high dose rates available with FFF beams make them ideal for high dose treatments, such as Volumetric Modulated Arc Therapy (VMAT)-delivery lung Stereotactic Ablative Radiotherapy (SABR), where treatment delivery is longer than that of standard treatments. They are also known to show reductions in treatment head scatter, multi-leaf collimator (MLC) transmission and treatment head leakage radiation, compared to flattened beams. The use of FFF beams for VMAT lung SABR has been shown to significantly reduce treatment delivery time, while maintaining plan quality and accuracy. Another potential advantage of the use of FFF beams for VMAT lung SABR is the reduction in peripheral (out-of-field) dose, due mainly to the reduction in head scatter and treatment head leakage. The peripheral doses delivered by VMAT Lung SABR treatments using 10X-FFF, 10X and 6X were investigated for the Varian TrueBeam medical linear accelerator. There were three components to this investigation; (1) Ion chamber measurement of peripheral dose for static open, static MLC and dynamic MLC fields, (2) Validation of Monte Carlo, Acuros XB and AAA algorithms for peripheral dose prediction, and (3) Evaluation of peripheral doses for VMAT lung SABR treatments using the validated Monte Carlo model. Measurements of out-of field doses for static open, static MLC and dynamic MLC fields showed that 10X-FFF delivered peripheral doses in the range of 30% to 50%, 3% to 40% and 5% to 20% lower than the peripheral doses for flattened beams. Dose calculation algorithm validation showed that AAA and Acuros XB significantly under predicted the dose in the peripheral region. Monte Carlo was found to be the most accurate dose calculation algorithm for peripheral dose prediction. The VMAT lung SABR dose distributions were calculated for both static gantry delivery and arc delivery using the validated Monte Carlo model. For static gantry Monte Carlo simulation, 10X-FFF was found to show a reduction in peripheral dose in the range of 7% to 21% and 7% to 17% when compared to 6X and 10X. For arc delivery Monte Carlo simulation, 10X-FFF was found to deliver a statistically significant reduction in mean peripheral dose compared to 6X in four of the six cases, and was not found to deliver a statistically significant reduction in mean peripheral dose compared to 10X in any of the six cases. For this type of VMAT lung SABR treatment, 10X-FFF offers a reduction in peripheral dose over 6X. In terms of the benefits of using 10X-FFF for this type of treatment, the reduction in peripheral dose is added to the already-established reduction in treatment times. / Graduate / 0756 / 0574

Overall, this study enhances our understanding of the role of c-Myc activation in chromosome 11 repositioning in mouse PreB v-abl/myc cells and a possible interaction between telomeres, TRF2, and lamin A/C underlying this phenomenon. Additionally, the importance of human 17q25.3 is confirmed as a potential region involved in NSCLC tumorigenesis. A utilizationof the 3D telomeric organization profiles is demonstrated a tendency to categorize NSCLC patients into different prognostic subgroups, underscoring a potential future value of its clinical application. / February 2017

To improve relatively poor outcomes for locally-advanced lung cancer patients, many current efforts are dedicated to minimizing uncertainties in radiotherapy. This enables the isotoxic delivery of escalated tumor doses, leading to better local tumor control. The current dissertation specifically addresses inter-fractional uncertainties resulting from patient setup variability. An automatic block-matching registration (BMR) algorithm is implemented and evaluated for the purpose of directly localizing advanced-stage lung tumors during image-guided radiation therapy. In this algorithm, small image sub-volumes, termed “blocks”, are automatically identified on the tumor surface in an initial planning computed tomography (CT) image. Each block is independently and automatically registered to daily images acquired immediately prior to each treatment fraction. To improve the accuracy and robustness of BMR, this algorithm incorporates multi-resolution pyramid registration, regularization with a median filter, and a new multiple-candidate-registrations technique. The result of block-matching is a sparse displacement vector field that models local tissue deformations near the tumor surface. The distribution of displacement vectors is aggregated to obtain the final tumor registration, corresponding to the treatment couch shift for patient setup correction. Compared to existing rigid and deformable registration algorithms, the final BMR algorithm significantly improves the overlap between target volumes from the planning CT and registered daily images. Furthermore, BMR results in the smallest treatment margins for the given study population. However, despite these improvements, large residual target localization errors were noted, indicating that purely rigid couch shifts cannot correct for all sources of inter-fractional variability. Further reductions in treatment uncertainties may require the combination of high-quality target localization and adaptive radiotherapy.

La progression tumorale est caractérisée par deux processus clés, la prolifération et l’invasion cellulaires. Les nAChRs, activés par la nicotine et ses nitrosamines dérivées (NNN et NNK), modulent les concentrations calciques intracellulaires et activent in vitro la prolifération, l’apoptose, la migration et l’invasion de lignées cellulaires tumorales. Dans cette étude, nous montrons, en utilisant des cultures primaires de cellules dérivées de cancers pulmonaires non à petites cellules (carcinomes épidermoïdes et adénocarcinomes), que les nAChRs α7 régulent différemment la prolifération cellulaire en fonction du stade de différenciation des tumeurs. Le nAChR α7 agit comme répresseur de la prolifération cellulaire dans les tumeurs bien différenciées et dans l’épithélium respiratoire normal, alors que dans les tumeurs peu différenciées, il stimule la prolifération cellulaire en réponse à la nicotine. A l’inverse, le nAChR α3α5β2 n’est que partiellement impliqué dans la régulation de la prolifération cellulaire aussi bien dans les tumeurs pulmonaires que dans l’épithélium respiratoire normal. Les nAChRs α7 et nAChRs α3α5β2 sont tous les deux impliqués dans la stimulation de l’invasion des cellules tumorales des carcinomes épidermoïdes et adénocarcinomes. Le polymorphisme non-synonyme rs16969968 de la sous-unité α5 induit une mutation au niveau d’un acide aminé hautement conservé (D398N). De nombreuses études d’association pangénomiques lient ce polymorphisme au développement des cancers pulmonaires. Dans cette étude nous montrons que les nAChRs exprimant la sous-unité α5 mutée (D398N) altèrent la prolifération et la différenciation des cellules respiratoires et modulent l’invasion des cellules tumorales, en synergie avec les nAChRs α7. / Tumor progression is characterized by two key processes, cell proliferation and invasion. Nicotinic receptors, activated by nicotine and its derived nitrosamines (NNK and NNN) modulate intracellular calcium concentrations and activate in vitro proliferation, apoptosis, migration and invasion of tumor cell lines. In this study, we show, by using primary cell cultures from lung cancer tumors, adenocarcinoma and squamous cell carcinoma, that nAChR α7 differently regulates cell proliferation according to the state of tumor differentiation. The α7 nAChRs acts as a repressor of cell proliferation in differentiated lung cancer tissues and in the normal respiratory epithelium, while it stimulates cell proliferation in response to nicotine, in poorly differentiated tumors. Conversely, the α3α5β2 nAChR is only partially involved in the regulation of cell proliferation in lung cancers and in the normal respiratory epithelium. The α7 and α3α5β2 nAChRs are both involved in the in vitro invasion process of adenocarcinoma and squamous cell carcinoma. Non-synonymous polymorphism rs16969968 in the CHRNA5 gene induces a mutation in a highly conserved amino acid (D398N). Many genome-wide association studies have demonstrated the relationship between this polymorphism and the incidence of lung cancer. In this study, we show that nAChRs, expressing the mutated α5 subunit (D398N), are in involved in the alteration of the proliferation and the differentiation state of respiratory epithelial cells, and also modulate tumor cell invasion, in synergy with the α7 nAChRs.

Le but de cette étude a été d’analyser l’intérêt de l’aérosolthérapie d’anticorps monoclonaux (AcM) dans le traitement du cancer broncho-pulmonaire non à petites cellules (CBNPC) en utilisant le cetuximab, un AcM anti-EGFR. L’expression du récepteur FcRn, essentiel dans la pharmacocinétique et la biodistribution des IgGs thérapeutiques a été étudiée dans le CBNPC. Nos résultats montrent que l’AcM peut résister aux contraintes physiques de la nébulisation. Les données de biodistribution et pharmacocinétique, obtenues dans un modèle animal, indiquent que l’Ac administré par aérosol, s’accumule de façon prolongée au niveau pulmonaire. De plus, les tumeurs chez la souris paraissent sensibles à l’aérosolthérapie avec l’AcM. Par ailleurs, le récepteur FcRn est moins exprimé au niveau transcriptionnel et protéique dans le tissu tumoral que dans le tissu sain adjacent à la tumeur dans le CBNPC. Cette altération de l’expression pourrait être due à l’hyperméthylation du promoteur de ce gène. / The project aims at determining whether aerosoltherapy is well suited for monoclonal antibodies (Mab), in Non-Small Cell Lung Cancer (NSCLC). In addition, exploration of the expression of FcRn, an IgG receptor contributing to increased half-life and biodistribution of Mab, has been studied in NSCLC. Using cetuximab, an anti-EGFR Mab, we showed that Mab resist the physical constraints of nebulization. Biodistribution and pharmacokinetic analyses, using a murine model, revealed that cetuximab is highly and durably accumulated within the lungs and slowly and weakly released into the bloodstream following airways delivery. Moreover, animal tumors seem sensitive to cetuximab aerosoltherapy. Expression analysis of FcRn at both the transcript and protein levels showed that the receptor is downregulated in NSCLC. FcRn alteration of expression in the tumor might be due to hypermethylation of the gene promoter as often found in cancer.

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. March 2015 / Lung cancer is currently the most deadly form of cancer due to the fact that metastasis occurs in the lymph nodes making it difficult to remove by surgical means. Chemotherapy has been the most successful method of treatment, although it has been harmful to human health as a consequence of non-specific cytotoxicity. There has been, therefore, a growing interest in cancer research to develop alternative cancer treatments, which are less toxic. Currently plant-derived drugs are perceived to be more effective as they display both cytotoxic activity and are less harmful to overall human health. Thus the aim of the study was to determine the cytotoxic effects of the plant Lobostemon fruticosus on A549 cells. The IC50 of the methanol and butanol extracts of L. fruticosus were obtained at 40 μg/ml and 50 μg/ml, respectively. DNA fragmentation was observed after 48 hour exposure to treatments, indicating that the plant extracts induced apoptosis. Cell cycle analysis indicated that the plant extracts inhibited cell cycle progression at the sub-G0 phase, which indicated that the cells had undergone apoptosis. RT-PCR showed that the expression of p53 was down-regulated; however, p21 and Bax were up-regulated in all treatments. LC-MS identified that the compounds from the plant extracts are known apoptotic inducers. The results lead to the conclusion that the extracts of L. fruticosus, induce cell death in A549 cells. The plant extracts induced a p53-independent apoptotic mechanism, which was mediated by Bax and p21. Key words: Lobostemon fruticosus, camptothecin, taxol, Non-small cell lung cancer (NSCLC)