学位論文内容の要旨（ Summary of dissertation）

博士の専攻分野の名称

博士（医 学）（Degree conferred: Doctor of Philosophy）

氏 名

ゴウダルジ・ホウマヌ（Name of recipient: Goudarzi, Houman）

学 位 論 文 題 名（Title of dissertation）
Enhancement of malignant properties of human malignant pleural mesothelioma
cells under hypoxia（低酸素環境下におけるヒト悪性胸膜中皮腫細胞の悪性化に関する研究）【Background and Objectives】
Malignant pleural mesothelioma (MPM) is an intractable tumor of mesothelial
cells lining the visceral and parietal pleura. Often MPM progresses to advanced
stages without clinical signs or symptoms, and has involved other organs by the timeof diagnosis; its prognosis is very poor even after multi-modality treatments.
Unfortunately MPM incidence is increasing and the peak of MPM mortality isapprehended to be in the near future. To date, there are only few pieces of informationof molecular mechanisms for development and malignant progression of MPM. Insolid tumors, the microenvironment often becomes hypoxic, a condition that theoxygen supply to tissues decreases, during the expansion of a tumor mass. It is known
that intratumoral hypoxia acts as both tumor-suppressive and –progressive factors.
In this study, I aimed to determine (a) whether hypoxia enhances malignant
behaviors such as cell growth, motility and invasiveness of MPM cells, and if it does,(b) by which mechanism(s) hypoxia involves the malignancy.
【Materials and Methods】
Six MPM cell lines including the 2 newly established cell lines (established inthis study) were used. These cell lines were cultured under hypoxia (1% O2) or
normoxia (21% O2) for 24-48 h. Cell growth was analyzed by assays for doubling time,colony and spheroid formation. Cell motility and invasiveness were analyzed byphagokinetic track assay and by an assay using type I collagen gel, respectively.
【Results】
Firstly, I examined whether the MPM cells showed adaptive cellular responses
to hypoxia. All the cell lines showed promoter activity through hypoxia-responsive
elements under hypoxia. Hypoxia upregulated the expressions of GLUT-1, VEGFA
and HK2, which are downstream genes of hypoxia-inducible factor (HIF). Hypoxia
reduced saturation density and sizes of spheroids whereas it enhanced colony-forming
ability. And hypoxia enhanced their cell motility and invasivenesss. Knockdown
analyses by using siRNA targeting HIF-1 and HIF-2

which are main transcription

factors responding to hypoxic stress, revealed that hypoxia-enhanced motility andinvasiveness were mediated through activation of HIF-1

but not HIF-2 . RT-PCR

analysis suggested that MUC1 gene which encoded a highly glycosylated membraneprotein could be a candidate of downstream genes of HIF-1 . To determine whether
HIF-1

activated MUC1 transcription, I performed promoter analysis by usingreporters with serial deletions of MUC1 promoter. As a result, it was found that apart (-1,473/-763 which contains two putative hypoxia-responsive elements (HRE)sites) of the promoter region was necessary for MUC1 transactivation under hypoxia.
Flow cytometric and immunoblot analyses revealed that hypoxia increased theexpression of sialylated MUC1 on the cell surface of MPM cells. To analyze the rolesof MUC1 in hypoxia-enhanced motility and invasiveness, I performed knockdown
experiments of the gene by using siRNAs, which reduced motility and invasiveness ofthe MPM cells under hypoxia.
【Discussion】
Thesefindings

oncellgrowthsuggestthecomplexityoftumor

microenvironment where additive factors such as changes of cell density alter theresponses of MPM cells to hypoxia. The results from the experiments of motility andinvasion indicated that the increased expression of MUC1 through activation ofHIF-1 pathway likely played an important role in the enhancement of cell motility
and invasiveness of MPM cells under hypoxia.
【Conclusion】
From these results, I conclude that hypoxia promoted in vitro malignant behaviors ofMPM cells, especially motility and invasiveness, through HIF-1 -MUC1 pathways.