京都大学 / 新制・課程博士 / 博士(医学) / 甲第24503号 / 医博第4945号 / 新制||医||1064(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 羽賀 博典, 教授 椛島 健治, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

According to the Centers for Disease Control, cancer is one of the leading causes of death in the United States. Characterized as a disease that develops as a result of an unstable genome, cancer is known to arise from numerous spontaneous mutations in the DNA of cells. Recent evidence shows that cancer cells within tumors are not self-reliant; rather, they progress along with other cells in their surrounding environment. Tumor cells recruit neighboring cells that, like the cancer cells, also become deregulated, forming the tumor stroma that aids in tumor progression. Within the stroma, cancer-associated fibroblasts (CAFs) play a vital role in the progression of cancer. Recent studies have found an important link between increased matrix stiffness surrounding the tumor and the invasion of the tumor. Thus, it is proposed that as the matrix stiffens, the tumor takes on more aggressive phenotypes. The transcriptional regulators YAP and TAZ (YAP/TAZ), key effectors of the Hippo pathway, are known to respond and influence matrix stiffening. In stiff matrix environments YAP/TAZ accumulate in the nucleus, and can drive transcriptional events. CAF's from late stage breast cancers have been found to exhibit increased YAP expression and increased ability to remodel and stiffen the extracellular matrix. Whether YAP or TAZ in these CAFs influences the metastatic properties of tumor cells is unclear. The present study aims to establish a link between YAP/TAZ activity in CAFs and cancer migration and invasion. We hypothesized that high nuclear activity of YAP/TAZ in fibroblasts would lead to non-autonomous signals that increase epithelial migration, and conversely that signals originating from epithelial cells affect YAP regulation in fibroblasts. We obtained CAFs from oral squamous cell carcinomas (OSCC) at various stages, and interestingly found that when CAFs obtained from stage III and stage IV tumors were co-cultured with OSCC cells they had the ability to cause OSCC cell migration. This CAF-induced migration was dependent on YAP/TAZ in the CAFs, as YAP/TAZ knockdown repressed this crosstalk. To gain insight into the mechanisms driving this process, transwell migration assays were conducted using NIH-3T3 fibroblasts engineered to overexpress YAP, or mutants of YAP, in doxycycline-inducible manner. We found that expression of YAP in NIH-3T3 cells, particularly a nuclear-localized YAP mutant, promoted the ability for OSCC cells to migrate in co-culture experiments. Media conditioned from these cells was sufficient to recapitulate this phenotype, suggesting that secreted factors from these fibroblasts may act as a signal that promotes migration. This activity of YAP was dependent on the ability for YAP to bind to the TEAD transcription factors, a major mediator of YAP transcriptional activity. Together these results indicate that nuclear YAP activity in fibroblasts can modulate the migration of neighboring cancer cells, suggesting that YAP plays a key role in stroma-cancer crosstalk during cancer progression.

Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...

Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18894号 / 医博第4005号 / 新制||医||1009(附属図書館) / 31845 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 戸井 雅和, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking β-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

<p>To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype. </p><p>In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.</p><p>In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated. </p><p>Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.</p> / Dissertation

iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

Abstract Squamous cell carcinoma (SCC) of the mobile tongue is the most common type of cancer of the oral cavity, accounting for 30-40% of oral cancers. It behaves aggressively and almost half of the affected patients still die of the disease despite great advances in its medical and surgical care. Ameloblastomas are the most common clinically significant type of odontogenic tumors, constituting approximately 1% of all cysts and tumors of the jaw. They are benign but locally invasive tumors with a strong tendency to recur after surgery. Ameloblastic carcinoma combines the histological features of ameloblastoma with cytologic atypia irrespective of the presence or absence of metastasis. The effectiveness of tight junction proteins (claudins 1, 4, 5, 7 and occludin) and cancer-associated fibroblasts (CAFs) as prognostic markers in OTSCC and as markers of malignancy in ameloblastomas was studied. Abundance of CAFs and Claudin 7 derangement was found to be associated with poor disease-specific survival in oral (mobile) tongue cancer. Appearance of CAFs within the epithelial islands of ameloblastoma was found to be a marker of malignancy in the tumor. The prognostic predictability of CAF density, Ki-67 (cell proliferation marker), maspin (tumor suppressor marker) and tumor DNA content (tumor ploidy using image cytometry) in tongue cancers was also tested. CAF density was the only marker strongly predictive of prognosis. In ameloblastomas, α-SMA (for CAFs), Ki-67, epithelial membrane antigen (EMA) and DNA content (using image and flow cytometry) were assessed as markers of ameloblastic carcinoma. Only α-SMA was able to predict ameloblastic carcinoma when found in the epithelial islands. In conclusion, staining for α-SMA and claudin 7 seems to be beneficial for prognostication in tongue cancer, while α-SMA staining may be beneficial in differentiating ameloblastoma from ameloblastic carcinoma.