京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18478号 / 農博第2078号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4862(農学部図書室) / 31356 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 小川 順, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18886号 / 医博第3997号 / 新制||医||1009(附属図書館) / 31837 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 戸口田 淳也, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Prostate cancer is the most commonly diagnosed cancer and the second most leading cause of cancer deaths in American men (www.cancer.org). Most prostate cancer-related deaths are due to the metastatic form of the disease. The 5-year relative survival rate in patient's diagnosed with metastatic prostate cancer is just 28%, as compared to 100% in patient's diagnosed with localized prostate cancer. This clearly indicates the lack of effective treatment available for metastatic prostate cancer. MicroRNAs (miRNAs) are small (18~23 nucleotide long) non-coding RNAs that can influence gene expression by binding to the 3'-untranslated region of coding RNAs at the post-transcriptional level. Some miRNAs has been termed as oncomirs due to their role in promoting tumor growth, invasion and metastasis. One such oncomir is microRNA-21 (miR-21) whose levels are often up-regulated in a number of cancers, including prostate cancer. MiR-21 increases the survival and invasiveness of cancer cells by suppressing its target tumor suppressor genes, namely programmed cell death 4 (PDCD4) and maspin. Thus, drugs which target miR-21 for inhibition could provide novel treatment options for metastatic prostate cancer. Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin found in high quantities in various dietary sources, such as grapes, red wine, berries and peanuts. Various reports have demonstrated a significant role of resveratrol in the management of several old age diseases including cancer. The efficacy of resveratrol as an anti-cancer agent resides in its ability to interfere with cell proliferation and metastasis and enhancement of apoptosis. Resveratrol has been shown to act on several intracellular targets to exert these effects. However, the exact mechanism by which resveratrol mediates its beneficial cancer chemotherapeutic actions are not clear and is the focus of this study. Based on the reported data, we hypothesized that resveratrol mediates its anti-cancer action against metastatic prostate cancer by inhibiting the signaling pathway which involves miR-21 expression and function. To address this hypothesis, we show that resveratrol decreased cell viability, migration and invasiveness of androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. These effects of resveratrol were associated with the inhibition of miR-21, since over-expression of miR- 21 with pre-miR-21 oligonucleotides attenuated resveratrol's effect on these cells. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21 and knockdown of PDCD4 by short interfering (si) RNA reversed the resveratrol's effect on prostate cancer cells. PC-3M-MM2 cells also exhibits high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002, a known PI3-kinase inhibitor. MiR-21 expression in these cells appears to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibits the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Future investigation into the molecular mechanisms revealed that resveratrol suppressed prostate cancer growth by decreasing the levels of insulin-like growth factor-1 (IGF-1) and its receptor (R). Previous studies had associated elevated levels of serum IGF-1 with high risk of prostate cancer. IGF-1, after binding to its receptors, acts as a potent mitogen which stimulates cancer cell growth and proliferation mainly by activating Akt signaling pathway. Interestingly, this effect of resveratrol on IGF-1/IGF-1R was independent of its effect on miR-21. In summary, our data show that resveratrol exerts its anti-cancer effect on metastatic prostate cancer cells, at least in part, by targeting Akt/miR-21 pathway. These data highlight a potential molecular mechanism for resveratrol's anti-cancer action for the treatment of metastatic prostate cancer and suggest that inhibition of the IGF-1/Akt/miR-21 pathway is a rationale approach for the treatment prostate cancer metastasis.

Although poorly understood, normal cells and cancerous cells of the same type exhibit different patterns of nutrient consumption, processing and utility of metabolic substrates. Differences in substrate uptake, preference, and alternately emphasized metabolic pathways offer opportunities for selective targeting of cancer versus stroma. This may be accomplished by using a sequential approach of nutrient deprivation and pharmaceutical perturbation of metabolic pathways to inhibit cellular proliferation. The purpose of this study was to investigate the effects of restricting glucose and glutamine concentrations, in vitro, to levels that resemble a potential human fasting state. The mammalian target of rapamycin (mTOR), a mediator of nutrient sensation, was then inhibited with rapamycin in the nutrient-restricted conditions. Because active Akt/mTOR is implicated in cancer cell pro-survival, the hypothesis is that pharmaceutical inhibition of active Akt/mTOR signaling in combination with the stress of restricted nutrient supply will be more effective than nutrient deprivation alone at disrupting metabolic processes to impair cancer cell proliferation and/or pro-survival mechanisms. Untreated and treated conditions were tested to determine if an additive or synergistic effect would result from a sequential insult of nutrient deprivation followed by inhibited mTORC1 signaling. The cell line used for this study was cultivated from a murine pancreatic intraepithelial neoplasia (PANIN) derived from a transgenic mouse with pancreatic tissue-specific expression of constitutively active Akt. The transgene of Akt, isoform 1, contains a myristoyl tag that facilitates co-localization of Akt to the plasma membrane, thereby promoting the activation of this signaling protein.; This aberrantly activated Akt represents a prosurvival condition observed in most cancers, and impacts metabolic balance with increased downstream signaling to metabolic sensors and regulators, including mTORC1. Several methods were used to evaluate changes in metabolic and physiological response to nutrient deprivation and mTORC1 inhibition. These included tetrazolium reduction/absorbance readings to qualitatively evaluate differences in cell proliferation, and Western immunoblots for observing changes in protein expression and phosphorylation. ATP luminescence assays were applied to quantify intracellular ATP content, and citrate synthase spectrophotometry used to quantify specific activity/indicate changes in the TCA/OXPHOS production of ATP. Results from the above methods suggest that, individually, nutrient deprivation and rapamycin treatment share some similar effects on metabolically-related protein phosphorylation and in reducing cellular proliferation. Collectively, nutrient deprivation plus rapamycin treatment, however, resulted in unanticipated metabolic alterations under conditions used for this study, the complexities of which would need to be delineated in future studies.

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Obesity-induced chronic inflammation plays a key role in the pathogenesis of insulin resistance and the metabolic syndrome. Proinflammatory cytokines can cause insulin resistance in adipose tissue, skeletal muscle and liver by inhibiting insulin signaling transduction. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a serine/threonine kinase functioning in Toll-like Receptor signaling pathways, and plays an important role in inflammation and immune response. In our studies, we demonstrated that IRAK-1 is involved with the negative regulation of PI3K-Akt dependent signaling pathway induced by insulin and TLR 2&4 agonists. Out data also indicate that IRAK-1 can interact with IRS-1 protein both in vivo and in vitro. The binding sites for the IRAK1-IRS1 biochemical interaction are IRS-1's PH domain and IRAK-1's proline-rich LWPPPP motif. Our studies also indicate that IRAK-1 is involved with the negative regulation of glycogen synthesis through inhibiting PI3K-Akt signaling pathway and thus releasing GSK3β's inhibitory effect on glycogen synthase. Moreover, our studies also suggest that IRAK-1 is involved in the activation of transcription factors CREB and ATF-1 by stimulating CREB-Ser133 and ATF-1 phosphorylation. CREB transcription factor family induces genes involved in cellular metabolism, gene transcription, cell cycle regulation, cell survival, as well as growth factor and cytokine genes. That may partially explain our finding that IRAK-1 may be also involved with cell proliferation and survival pathway. / Master of Science