京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20995号 / 医博第4341号 / 新制||医||1027(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 武藤 学, 教授 川口 義弥, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24790号 / 医博第4982号 / 新制||医||1066(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 竹内 理, 教授 斎藤 通紀, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.

BACKGROUND:The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion.RESULTS:We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting.CONCLUSION:We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.

Hyaluronan is a ubiquitous glycosaminoglycan which is an important constituent of the extracellular matrix (ECM). In addition to organizing the extracellular matrix and regulating tissue homeostasis, hyaluronan, by binding to its main cell surface receptor CD44, is involved in intracellular signaling pathways regulating major cellular processes during development, wound healing, inflammation and cancer. Accumulation of hyaluronan in cancer promotes progression of the disease and correlates with poor prognosis. This thesis focuses on the regulation of hyaluronan synthesis and its signaling in normal and cancer cells. Cancer cells in solid tumors are surrounded by stroma, which has an essential role in the growth and metastasis of tumors. Prominent members of the tumor stroma are fibroblasts, which synthesize ECM components, such as hyaluronan, and secrete growth factors, and activate intracellular signaling pathways. We demonstrate a cross-talk between the receptors for platelet-derived growth factor BB (PDGF-BB), transforming growth factor β (TGFβ) and CD44 in dermal fibroblasts. We found that PDGF-BB can activate the Smad signaling pathway downstream of the TGFβ receptor I (TβRI), and that PDGF-BB-induced migration depends on TβRI. CD44 forms a ternary complex with the receptors for PDGF-BB and TGFβ, and negatively regulates their signaling. Furthermore, we demonstrate that TGFβ stimulation of mammary epithelial cells transcriptionally upregulates hyaluronan synthase 2 (HAS2), which is essential for TGFβ-induced epithelial-mesenchymal transition (EMT); in this process, polarized epithelial cells adapt a mesenchymal phenotype which facilitates migration and invasion. HAS2 protein activity and stability is regulated by posttranslational modifications, including ubiquitination. We investigated the ubiquitination of HAS2 in aggressive breast cancer cells, whose metastasizing capability depends on HAS2-synthesized hyaluronan. We identified two deubiquitinating enzymes, USP4 and USP17, which target HAS2 and affect its activity and stability. In summary, these studies increase the knowledge about the regulation of hyaluronan production and its role in cancer progression.

Prostate cancer (PCa) cells predominantly metastasize to bone and the complex crosstalk between PCa cells and osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells) leads to increased tumour growth and worsening of bone disease. Understanding the mechanisms of PCa bone metastasis can identify the aggressive fraction of PCa resulting in earlier intervention. The ability of PCa cells to express bone cell-specific features, termed osteomimicry, could potentially explain the osteotropic nature of PCa cells. The aim of this study was to determine the role of osteomimicry in the regulation of epithelial-mesenchymal transition (EMT) in bone metastatic prostate cancer cells. It was demonstrated that the osteoblast-specific marker alkaline phosphatase (bone/liver/kidney) (ALPL) was overexpressed in bone metastatic (ARCaPM), compared to non-metastatic (ARCaPE), human PCa cells. Knockdown of ALPL resulted in decreased cell viability, increased cell death and a change from mesenchymal to epithelial morphology in ARCaPM and PC3 cells, and increased CDH1 expression along with decreased migration in ARCaPM cells. Treatment with extracellular ATP also resulted in decreased viability, increased expression of epithelial markers (CDH1, KRT14) and decreased expression of mesenchymal markers (VIM, ZEB1), and reduced expression of ALPL in ARCaPM cells. Small RNA-sequencing identified microRNAs differentially expressed between ARCaPE and ARCaPM PCa cell lines: miR-373 expression was lower in ARCaPM compared to ARCaPE cells and its overexpression in ARCaPM cells resulted in a change to epithelial morphology, increased expression of the epithelial marker CDH1 and decreased expression of the mesenchymal markers VIM and ZEB1. Finally, the development of a high-throughput screening method to identify novel microRNA regulators of osteomimicry was described, which identified two microRNAs miR-199a-5p and miR-212 as positive regulators of ALP activity. Taken together, this thesis describes the identification of ALPL and ATP as novel regulators of epithelial-mesenchymal transition in PCa cells and high-throughput ALP-activity screening as a powerful tool to identify novel microRNA regulators of ALP expression.

Diferentes estudos têm demonstrado uma variação importante na frequência diagnóstica dos tumores de glândula hepatoide em cães, possivelmente devido à ausência de critérios morfológicos claros e bem estabelecidos em seu diagnóstico. Não estão bem definidos os critérios clínico-patológicos associados ao comportamento biológico destas neoplasias. Alguns estudos levantam a hipótese de participação do mecanismo de transição epitélio-mesenquimal devido à frequente imunoexpressão de vimentina em tumores de glândula hepatoide, porém esta hipótese permanece sem comprovação. O objetivo deste trabalho foi avaliar a concordância diagnóstica entre patologistas, assim como investigar o mecanismo de transição epitélio-mesenquimal e a taxa de proliferação celular por marcadores imuno-histoquímicos e, por fim, correlacioná-los com sobrevida para investigação de possíveis valores prognósticos. Cinco patologistas avaliaram 57 tumores de glândula hepatoide quanto ao diagnóstico e quanto a diversos critérios morfológicos e, com base nestes critérios, foram desenvolvidos algoritmos diagnósticos por meio de árvores de regressão por partição recursiva. Adicionalmente, foram investigadas as imunoexpressões de vimentina, pancitoqueratina, E-caderina e Ki-67 em 78 lesões, comparando-as entre glândulas normais, tumores benignos, tumores malignos e tumores malignos vasculoinvasivos/metastáticos. A sobrevida livre de recorrência local, recorrência da doença, sobrevida global e sobrevida doença-específica foram investigadas em 33 pacientes. Houve concordância média razoável em análise interobservador quanto ao diagnóstico (k=0,54), com potencial de melhora para concordância média boa após aplicação de dois novos algoritmos diagnósticos (k=0,64 e 0,65, respectivamente). Em relação aos marcadores imuno-histoquímicos, foram observadas maior expressão de vimentina e menor expressão de E-caderina em tumores malignos em comparação aos tumores benignos. Adicionalmente, observou-se uma forte correlação negativa entre a expressão de vimentina em células de reserva e expressão de pancitoqueratina em células diferenciadas (p=-0,65, p<0,0001), além de maiores taxas de proliferação celular (Ki-67) e contagem de mitoses em tumores malignos em comparação com tumores benignos. Em análise univariada de sobrevida doença-específica, os fatores prognósticos foram a presença de invasão vascular ou metástase no momento do diagnóstico, o escore de E-caderina em células de reserva e diferenciadas, e o tamanho tumoral. Em relação ao tempo de sobrevida livre de doença, os fatores prognósticos foram a presença de invasão vascular ou metástase no momento do diagnóstico, escore de E-caderina em células de reserva e diferenciadas, perda de polaridade celular, grau de infiltração, histórico de doença recorrente, escore de morfologia fusiforme e proporção de células de reserva. Os resultados do presente estudo confirmaram que existe um grau de discordância diagnóstica entre patologistas que poderia explicar parte da variação observada na prevalência de carcinomas de glândula hepatoide em literatura, e de que existe potencial para melhora da concordância diagnóstica por meio dos algoritmos propostos. Ainda, os achados reforçaram a hipótese de que o mecanismo de transição epitélio-mesenquimal participa na progressão tumoral em neoplasias hepatoides em cães, explicando a frequente imunoexpressão de vimentina observada nestes tumores. Por fim, foi demonstrado que a presença de invasão vascular ou metástase no momento do diagnóstico tem impacto significativo na sobrevida dos animais, assim como há outras características clínicas, morfológicas e imuno-histoquímicas com valor prognóstico para recorrência da doença e sobrevida. / An important variation in the prevalence of canine hepatoid gland carcinoma is currently reported, likely due to lack of consistent and well-defined criteria. Additionally, clinical and pathological features predictive of biological behavior are not well defined. The epithelial-mesenchymal transition mechanism has been proposed as a possible explanation for the frequent expression of vimentin by neoplastic hepatoid gland cells, but this hypothesis remains elusive. The present study aimed to define the level of diagnostic agreement between different pathologists, investigate immunohistochemical and morphological features indicative of epithelial-mesenchymal transition, and to evaluate candidate features that might be predictive of biological behavior. Five pathologists diagnosed and classified morphological features for 57 hepatoid gland tumors, and diagnostic algorithms were built with recursive partitioning analysis. Additionally, vimentin, pancytokeratin, E-cadherin and ki-67 immunohistochemical expression were evaluated for 78 lesions, and expression levels were compared between normal glands, benign tumors, malignant tumors, and vasculo-invasive/metastatic tumors. Local recurrence free survival, disease free survival, overall survival and disease-specific survival were evaluated for 33 cases. There was a fair mean interobserver agreement for diagnosis (k=0,54), that could potentially be improved to a good mean agreement with the use of two novel diagnostic algorithms (k=0,64 and 0,65). Additionally, higher levels of vimentin expression and lower levels of E-cadherin expression were noted in malignant tumors, as compared to benign tumors, and there was a strong negative correlation between vimentin expression in reserve cells and pancytokeratin expression in differentiated cells (p=-0,65, p<0,0001). Proliferation index (ki67) and mitotic count were higher for malignant tumors when compared to benign tumors. In univariate survival analysis, vascular invasion or metastasis at the time of diagnosis, E-cadherin score in differentiated and reserve cells and tumor size were predictive of disease-specific survival. Vascular invasion or metastasis at the time of diagnosis, E-cadherin score in differentiated and reserve cells, loss of cell polarity, grade of invasiveness, history of recurrent disease, spindle cell morphology score and proportion of reserve cells were predictive of disease-free survival. The results of this study demonstrate a level of diagnostic disagreement between pathologists that might explain, at least partially, the variation of prevalence of hepatoid gland carcinoma in current literature, and that this diagnostic agreement could potentially be improved. Also, these findings support the role of epithelial-mesenchymal transition in the progression of canine hepatoid gland tumors, and this mechanism could explain the frequent expression of vimentin in this particular neoplasia. Finally, it was shown that vascular invasion or metastasis at the time of diagnosis has a significant impact on survival, and that additional clinical, morphological and immunohistochemical features might also have predictive value for disease recurrence and survival.

A ação metastática do BPV permanece não clara. Este estudo avaliou a ação do BPV na transição epitélio-mesênquima (TEM), empregando linhagens celulares de papiloma (P), fibropapiloma (FB) e carcinoma de esôfago (CE). Os resultados mostraram a presença de infecção produtiva e o aumento do potencial proliferativo nestas células. Porém, foi verificada a redução do potencial de membrana mitocondrial em relação à pele saudável (controle) e o aumento do estresse oxidativo, resultante da ação da oncoproteína E6, justificando a clastogenicidade e a aquisição do fenótipo-tronco descrito nas linhagens de P, FB e CE. Estas linhagens mostram capacidade migratória decorrente do sequestro citoplasmático de E-caderina, e o aumento dos níveis de expressão de vimentina, vinculina e N-caderina como consequência da ativação dos fatores STAT3 e SLUG, sugerindo a ação do vírus na indução da TEM. Tais resultados foram validados em amostras de tecido, reforçando a ação do vírus na TEM, bem como demostrando o potencial destas linhagens celulares como modelo de estudo da metástase. / BPV metastatic action remains unclear. This study evaluated the BPV action on epithelial-mesenchymal transmition (EMT), using cell lines form papilloma (P), fibropapilloma (FB) and esophageal carcinoma (EC). Results showed the productive infection presence and the proliferative potential increase in these cells. However, it was verified the mitochondrial membrane potential loss in relation to normal skin (control) and the oxidative stress increase as result of E6 oncoprotein, justifying the clastogenicity and stem-cell phenotype acquisition described in P, FB and EC cells. This cell lines showed a migratory capability as result of cytoplasmic sequester of E-cadherin, and the increase levels of vimentin, vinculin and N-cadherin as consequence of STAT3 and SLUG factors activation, suggest the virus action on EMT. These results were also verified in tissue samples, reinforcing the BPV action on EMT, as well as demonstrating the potential of these cell lines as model to study the metastasis.