京都大学 / 新制・課程博士 / 博士(医学) / 甲第23607号 / 医博第4794号 / 新制||医||1055(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 溝脇 尚志, 教授 木村 剛, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with a poor prognosis. Currently, prognosis for HCC patients remains poor as few therapies are available. The clinical need for more effective HCC treatments remains unmet partially because HCC is genetically heterogeneous and HCC driver genes amenable to targeted therapy are largely unknown. Mutations in the TP53 gene are found in ~30% of HCC patients and confer poor prognosis to patients. Identifying genes whose depletion can inhibit HCC growth, and determining the mechanisms involved, will aid the development of targeted therapies for HCC patients. Therefore, the first half of this thesis focuses on identifying genes that are required for cell growth in HCC independent of p53 status. We performed a kinome-wide CRISPR screen to identify genes required for cell growth in three HCC cell lines: HepG2 (p53 wild-type), Huh7 (p53-mutant) and Hep3B (p53-null) cells. The kinome screen identified 31 genes that were required for cell growth in 3 HCC cell lines independent of TP53 status. Among the 31 genes, 8 genes were highly expressed in HCC compared to normal tissue and increased expression was associated with poor survival in HCC patients. We focused on TRRAP, a co-factor for histone acetyltransferases. TRRAP function has not been previously characterized in HCC. CRISPR/Cas9 mediated depletion of TRRAP reduced cell growth and colony formation in all three cell lines. Moreover, depletion of TRRAP reduced its histone acetyltransferase co-factors KAT2A and KAT5 at the protein level with no change at the mRNA level. I found that depletion of KAT5, but not KAT2A, reduced cell growth. Notably, inhibition of proteasome- and lysosome-mediated degradation failed to rescue protein levels of KAT2A and KAT5 in the absence of TRRAP. Moreover, tumor initiation in an HCC mouse model failed after CRISPR/Cas9 depletion of TRRAP due to clearance via macrophages and HCC cells depleted of TRRAP and KAT5 failed to grow as subcutaneous xenografts in vivo. RNA-seq and bioinformatic analysis of HCC patient samples revealed that TRRAP positively regulates expression of genes that are involved in mitotic progression. In HCC, this subset of genes is clinically relevant as they are overexpressed compared to normal tissue and high expression confers poor survival to patients. I identified TOP2A as one of the mitotic gene targets of the TRRAP/KAT5 complex whose inhibition greatly reduces proliferation of HCC cells. Given that this was the first time the TRRAP/KAT5 complex has been identified as a therapeutic target in HCC, the second half of this thesis focuses on identifying the mechanism via which depletion of this complex inhibits proliferation of HCC cells. I discovered that depletion of TRRAP, KAT5 and TOP2A reduced proliferation of HCC cells by inducing senescence. Typically, senescence is an irreversible state of cell cycle arrest at G1 that is due to activation of p53/p21 expression, phosphorylation of RB, and DNA damage. Surprisingly, induction of senescence after loss of TRRAP, KAT5 and TOP2A arrested cells during G2/M and senescence was independent of p53, p21, RB and DNA damage. In summary, this thesis identifies TRRAP as a potential oncogene in HCC. I identified a network of genes regulated by TRRAP and its-cofactor KAT5 that promote mitotic progression. Moreover, I demonstrated that disruption of TRRAP/KAT5 and its downstream target gene TOP2A result in senescence of HCC cells independent of p53 status. Taken together, this work suggests that targeting the TRRAP/KAT5 complex and its network of target genes is a potential therapeutic strategy for HCC patients.

As we face an expanding elderly population and increased age-related chronic diseases, we must seek solutions that address interconnected problems. Dasatinib plus quercetin (D+Q), a novel combination drug, is a proposed solution to improve the health and longevity of aged individuals with minimal side effects, exceptional efficacy, and maximum convenience. In this experiment, intermittent D+Q treatment was completed on young and old mice. RNA was extracted from perigonadal adipose tissue and cDNA libraries of mRNA and miRNA were made. Real-time quantitative polymerase chain reaction was used to assess relative gene expression for mRNA and miRNA pertaining to cell senescence, inflammation, and insulin resistance. miR-146a was found to have a significant difference (p < 0.05) in relative expression based on the interaction between age and treatment, while other genes showed no difference in relative expression. Furthermore, miR-149 was found to show near significance in interaction (p=0.0552) and had significant difference between old control and old D+Q treated mice. The miR-149 presents an avenue for future research in senescence markers and shows potential as a novel senolytic target.

Bone is a metabolically active tissue that is continuously remodeled throughout life. Osteocytes, the most abundant cells in bone, regulate bone homeostasis in response to hormonal and mechanical cues. Parathyroid hormone (PTH), a calciotropic hormone secreted from the parathyroid glands, has been widely used in the clinic to treat age-related osteoporosis. PTH acts on cells of the osteoblast lineage, including osteocytes, by signaling via the PTH receptor (PPR) to promote bone formation. However, the role of PPR signaling in osteocytes during aging has not been investigated. The hypothesis of this study is that PPR signaling in osteocytes plays a key role in maintaining skeletal health in aging mice. To address this hypothesis, mice in which the PPR was ablated in mature osteoblasts/osteocytes (Dmp1-Cre+;PPRfl/fl or Dmp1-PPRKO) were used to study their skeletal phenotype at 4 and 13 months of age. Compared to control littermates (Dmp1-Cre–;PPRfl/fl), Dmp1-PPRKO animals displayed age-dependent osteopenia due to reduced osteoblast activity and increased osteoclast numbers and activity. These changes were associated with a significant decrease in osteoprogenitors and an increase in marrow adiposity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes was accompanied by a marked increase in serum sclerostin, RANKL-expressing marrow adipocytes, and early onset of oxidative stress in osteocytes. In vitro studies demonstrated that PTH protected osteocytes from oxidative stress-induced cell death by suppressing the intracellular accumulation of reactive oxygen species. Mechanical forces are also important regulators of bone mass and quality. For instance, immobilization and reduced mechanical loading, such as prolonged bed rest or long-duration spaceflight, lead to bone loss or osteopenia due to reduced bone formation and increased bone resorption. Osteocytes are known to sense and transduce mechanical forces applied to the skeleton into biochemical signals. However, the exact molecular mechanism remains unclear. To unravel the mechanism by which osteocytes sense and respond to mechanical unloading, an osteocytic cell line, Ocy454, was exposed to microgravity (µG) conditions for 2, 4, or 6 days onboard the SpaceX Dragon-6 and the International Space Station. Global transcriptomic analysis demonstrated that µG leads to downregulation of key osteocytic marker genes compared to ground controls (1G), suggesting the impaired differentiation of osteocytes. Importantly, glycolysis was the most activated signaling pathway in osteocytes subjected to µG compared to 1G. Gene comparison analysis further identified a set of mechano-sensitive genes that are consistently regulated in multiple types of cells exposed to µG, suggesting a common, yet to be fully elucidated, genome-wide response to µG. In summary, these studies demonstrated that osteocytes are highly regulated by PTH and mechanical forces. We found that PPR signaling in osteocytes is important for protecting the skeleton from age-induced osteopenia by promoting osteoblast’s bone-forming activity and mitigating osteoclast’s bone resorption. We also demonstrated that PTH protects osteocytes from oxidative stress. Finally, we showed that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23799号 / 医博第4845号 / 新制||医||1058(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 妹尾 浩, 教授 中川 一路, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Serial passaging of primary human fibroblasts leads to the formation of non-dividing senescent cells by a process termed replicative senescence. This tissue culture-based methodology is currently used as a model system to determine the underlying mechanisms of in vivo cellular aging and tumor suppression. Senescence is regarded as an alternative pathway to apoptosis, where cells undergo multiple changes in metabolic and cellular signaling pathways in order to prevent proliferation but still maintain a metabolically-active cell. Whether or not this model accurately reflects in vivo processes is presently controversial; however, replicative senescence is currently the most applicable model through which one can investigate the underlying causes of human cellular aging in the context of controlled environmental stress over time. This work was directed at understanding the molecular processes involved in replicative senescence with specific emphasis on the role of the mitochondria. A series of experiments were performed to assess changes during the induction of replicative senescence under conditions of low (3%) and high (20%) oxygen levels. Measurements were made at the transcriptional, protein, and metabolite levels. Microscopy wasalso utilized to monitor changes in mitochondrial morphology and volume. While previous studies have evaluated specific pathways and/or products; this work combines a more complete metabolomic, genomic, proteomic, and morphological picture of cells undergoing senescence and oxidative stress. Considering the low cell population densities of primary adherent fibroblasts and the subsequent low concentrations of small polar metabolites involved in glycolysis and the TCA cycle, methodologies needed to be developed in order to optimize metabolite extraction and liquid chromatography-mass spectrometric analysis. Protein kinase and transcriptional microarrays were also performed in order to quantify the changes in activated/deactivated signaling cascades as well as gene expression and relate these findings to metabolomic data. Mitochondrial dynamics of cells at different age time points and under different oxygen conditions were also assessed including mitochondrial size, shape, membrane potential, and percent volume per cell volume using confocal microscopy. The results obtained not only confirm the major pathways involved in senescence (p53/p21, PTEN/p27, and RTK/Raf/MAPK) but also provide evidence at both the transcriptional and protein levels for additional senescence-associated pathways. The majority of the changes observed were related to pathways involved in cellular stress, cell cycle control, and the survival response. Metabolic data suggested a –pooling effect– of glycolysis and TCA precursor molecules due to attenuation in enzyme function; this theory was also supported by an observed up regulation of gene expression as a compensatory mechanism. Mitochondria exhibited changes in membrane potential as well as volume and percent volume per cell which suggested compensatory hypertrophy and/or attenuation of mitochondrial fission processes. When the aforementioned analyses are tied together, a “theoretical model of senescence” can be formulated and is characterized by increased metabolic protein and associated metabolite levels due to attenuation in their respective enzyme function, resulting in increases in expression of their associated genes as a compensatory mechanism. / Ph. D.