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Dlk is a transmembrane protein that possesses six epidermal growth factor-like sequences at the extracellular domain, a single transmembrane domain and an intracellular tail. The extracellular EFG-like region of Dlk can be released by action of an unknown protease that cuts the extracellular region near the cell membrane. Dlk belongs to the EGF-like homeotic protein family and has received many names: pG2, FA-1, Pref-1, SCP-1, ZOG and Dlk. All the proteins are identical or polymorphic products of a single gene. Dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and neuroendocrine differentiation. Dlk is also known as the preadipocyte factor-1 (Pref-1), is highly expressed in preadipocytes but is completely abolished in adipocytes. Pref-1 may function in the maintenance of the preadipocyte state and is a negative regulator of adipocyte differentiation. Dlk is expressed in tumors with neuroendocrine features, such as human neuroblastoma, rat pheochromocytoma, and a subset of Small Cell Lung Cancer (SCLC) cell lines. The Dlk expression is probably associated with some differentiation stages because the most undifferentiated cells were lacking expression of Dlk. The finding suggests that Dlk plays an important role in differentiation and tumorigenesis of several cell types. The study was designed to examine the influence of dlk overexpression on tumorigenicity of hepatoma cells. We constructed the mammalian expression vectors for full-length dlk, dlk extracellular domain, which were transfected into SK-Hep-1 cells for generation of stable clones. The transgene expressions in selected stable clones were verified by QRT-PCR and western blot analysis. Our results indicated that overexpression of extracellular domain significantly promoted the viability of SK-Hep1 cells during serum deprivation. In SCID mice, injection of full-length dlk clones led to increased tumor growth compared with the control groups. However, the migration ability was reduced in Dlk stable clones. In summary, these results suggested full-length Dlk promoted the tumor growth but reduced the migration ability of SK-Hep1 cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Hepatocarcinogenesis is considered a multifactorial and mulitstep process that involves the activation of oncogenes or the inactivation of tumor suppressor genes. Tumor suppressor gene PTEN (also known as MMAC or TEP1) is located on human chromosome 10q23. The 403¡Vamino acid PTEN protein encodes dual specificity protein phosphatases. Mutation of the PTEN is a common event in advanced stage of diverse human cancers. In our previous studies, immunohistochemical analysis indicated that reduced PTEN expression was found in nearly 40% of HCC specimens. Furthermore, restored PTEN expression by adenovirus gene delivery effectively inhibited the in vitro and in vivo tumorigenicity of Mahlavu cells, a human HCC cell line with PTEN inactivation. In the present study, we further characterize whether PTEN gene delivery still suppressed the oncogenic potential in HCC cell lines with functional PTEN. By expression and sequencing analysis, we identified human SK-Hep-1 cells as the hepatoma cell line with functional PTEN expression. The optimal condition for adenovirus vector to infect SK-Hep-1 cells was determined at the multiplicity of infection (MOI) of 50-100. Tough SK-Hep-1 cells were effectively transduced with exogenous PTEN gene, the enhanced PTEN expression by adenovirus gene delivery did not alter the phosphoryation extent of Akt in SK-Hep1 cells. Nevertheless, PTEN gene delivery reduced the proliferation of SK-Hep-1 cells by ~20%. In addition, the motility of PTEN-transduced SK-Hep-1 cells significantly decreased comparing to cells of control groups. Western blot analysis suggested the decreased cell motility might be attributed to the reduced phosphorylation of focal adhesion kinase (FAK) by PTEN gene delivery. Above all, PTEN gene delivery profoundly reduced the colony formation of SK-Hep-1 cells in soft-agar. However, PTEN gene delivery did not affect the secretion of matrix metallo-proteinases (MMPs) release. Animal studies will be carried out in the future to validate the present in vitro findings. In summary, PTEN gene delivery holds promise for treatment of HCC even when the hepatoma cells possess functional PTEN gene.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13518号 / 論医科博第10号 / 新制||医科||10(附属図書館) / (主査)教授 金子 新, 教授 松田 秀一, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Wilms' tumor 1 gene is overexpressed in many types of cancer and is associated with poor prognosis and resistance to anti-cancer therapies. In vitro studies in non-glioma cells types have demonstrated that WTl plays a role in increased proliferation, resistance to apoptosis, and increased cellular invasion. We aimed to thoroughly characterize the expression pattern of Wilms' tumor 1 in human malignant glioma and discern its function in this complex disease process. We screened a large sample of established human malignant glioma cell lines and glioma tissue specimens of all grades for WT1 expression. The majority of cell lines and 80% of all glioma tissue expressed WTI mRNA, all of which expressed WTl(+KTS) isoforms. Further screening of the glioblastoma specimens for p53 mutation followed by logistic regression analysis demonstrated a positive correlation between WTl expression and wild-type p53 (p = 0.04). To determine if WTl and p53 functionally interacted, we generated LN-229 glioblastoma cells that stably expressed WTl. As LN-229 cells harbor a p53 mutation, transient transfection with wild-type p53 induced apoptosis. However, stable WTI expression did not protect cells from p53-mediated cell death. We then generated U87MG cells (p53 wild-type) that stably expressed WT1 to model an endogenous p53 response. It is well known that after treatment with ionizing radiation, U87MG cells readily undergo p53-mediated apoptosis. Again, WTI expression did not protect against ionizing radiation induced p53-mediated cell death. We next examined the effect of transient WTI silencing on ionizing radiation induced cell death in T98G and LN-18 cells which express endogenous WTl. Combination treatment with ionizing radiation and silencing of WTI using short interfering RNA caused a decrease in viability and clonogenic survival relative to radiation alone in both cell lines. Lastly, we studied the effect of stable WTl silencing using short hairpin RNA on glioblastoma cell tumorigenicity. Stable transduction of U25 1MG and LN-18 cells with WTI short hairpin RNA resulted in a marked decrease in proliferation. WTI silencing in U251MG cells also caused a decrease in in vitro invasion. WTl silencing in U251MG cells caused an increase in tumor latency and a decrease in tumor growth rate when cells were used to subcutaneously inoculate nude mice. Not only do these studies support an oncogenic role for WTI in glioma biology, they provide encouraging evidence that WTl may be a therapeutic target for molecular treatment of glioblastoma.

Background: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair.

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