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1 学位論文の内容の要旨論文提出者氏名赵鑫論文審査担当者主査原田清副査髙野吉郎原田浩之論文題目 Aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological featuresof oral squamous cell carcinoma ( 論文内容の要旨 ) Abstract Disruption of cell adhesion plays a pivotal role in dedifferentiation, invasion and metastasis of various cancers. The desmosome and hemidesmosome are anchoring junctions that control cell-cell and cell-matrix adhesion respectively. To clarify their contributions in mediating the biological properties of oral cancer, we immunohistochemically examined the expression of desmoglein 1 (DSG1), DSG2, DSG3, desmocollin 2 (DSC2), bullous pemphigoid 180 (BP180), Laminin 332 in 51 cases of oral squamous cell carcinoma. DSG1, DSG3 and DSC2 were expressed in normal oral epithelium, showing localization on the plasma membranes, although DSG1 expression was almost absent in the basal layer. In cancer, their membranous expression reduced showing a cytoplasmic translocation. BP180 expression was observed on the plasma membranes of the basal cells. In cancer, BP180 was observed at the tumor-stroma interface, often showing cytoplasmic staining. Laminin 332 was absent in normal epithelium but its expression was consistently induced in the cancer cells surfacing the stroma. We sub-quantitatively evaluated the expression by evaluating the membrane staining and correlated them to the clinicpathological parameters including the histologic differentiation, pattern of invasion and lymph node metastasis. Decrease of DSG3 and DSC2 expression correlated to less differentiated and more invasive histologic features, and higher incidence of lymph node metastasis. Decrease of BP180 and increase of Laminin 332 expression were also associated with these undesirable cancer phenotypes. These results demonstrated the significance of desmosomal and hemidesmosomal proteins in generation of each oral cancer property. Combined use of these markers may prove useful for grading cancers and predicting the prognosis. Introduction Desmogleins (DSGs) and desmocollins (DSCs); transmembrane proteins that belong to the - 1 -

2 cadherin superfamily, constitute desmosomes and homophilically interact between the extracellular domains of adjacent cells. Hemidesmosomes employ integrin 4 for the major constituents to connect the basal surface of an epithelial cell to the basement membrane that is composed of the secreted proteins such as laminin 332. Bullous pemphigoid 180 (BP180) and BP230. The expression of DSG2, DSG3, is increased compared with normal tissue in certain cancers of the skin, head and neck, prostate and lung, and this increased expression is associated with enhanced tumour progression and reduced patient survival. By contrast, the loss or reduction of DSG1 3, DSC2, DSC3, is observed on the development and the progression of various human cancers, including skin, head and neck, gastric, colorectal and breast cancers, often correlating with advanced tumour grade, increased metastasis and poor prognosis. Loss of hemidesmosome is documented by electron microscopy in cutaneous basal cell carcinoma and cultured breast cancer cells exhibits reduced abilities of hemidesmosome formation compared to the normal breast epithelial cells, and the expression of hemidesmosome components such as integrin 4 BP180 and BP230 is reduced or mislocalized. Although integrin 4 is either highly expressed or downregulated in various cancers, the alteration of integrin 4-mediated signaling is thought to play vital roles in the biology of cancer. In this study, we tried to extend these observations, in particular, to get an insight into the possible coordinated regulation of the desmosomal and hemidesmosomal components, and its disparity in oral squamous cell carcinoma. Materials and methods Tissue microarray construction Over 200 oral squamous cell carcinoma cases in the archives of Dental Hospital of Tokyo Medical and Dental University were assessed whether they were appropriate for tissue microarray. We conducted Ki67 and p53 immunostaining on randomly selected 20 cases and confirmed the immunostainability. Eventually 60 cases were selected as appropriate for tissue microarray and representative areas of them were marked on HE stained sections in a 5 mm-diameter circle, and the corresponding cylinders were punched from selected areas of paraffin blocks to create 3 pieces of 20-sample tissue multiblocks. Immunohistochemistry Four-micrometer thick sections were cut and placed on silane-coated slides. Heat-induced antigen retrieval was performed in 10 mm Tris (ph9.0)/1mm EDTA for 5 minutes using autoclave pot at 120ºC. Proteinase K (20 ug/ml) treatment was conducted for 10 min at room temperature. Endogenous peroxidase was blocked using 3% hydrogen peroxide solution for 15 min. EnVision+Dual Link was used for the secondary antibody reaction. 3,3'-Diaminobenzidine was used for the chromogenic substrate. Connective tissues and lymphocytes in the specimens served as an internal negative control

3 Results DSG1 was expressed in the oral epithelium, epidermis and epithelial appendages. DSG1 exhibited membranous staining in most parts, but cytoplasmic staining was observed in ductal cells. DSG2 was weakly expressed showing a diffuse cytoplasmic pattern in the epithelial appendages but was not detected in the epidermis and oral epithelium. DSG3 was strongly expressed showing a membranous pattern in the oral epithelium, hair follicles and ducts of sweat glands. BP180 was expressed in the basal cells of the oral epithelium, epidermis and epidermal appendages showing a membranous pattern, with weak cytoplasmic staining in the epidermis. The scores for the expression of DSG1, DSG3 and DSC2 were reduced compared to the normal epithelium in most cancers. DSG2 was almost consistently induced in the oral cancer, although the expression was weak and it was with a cytoplasmic pattern, and the membranous pattern was not observed in any cases. The scores of DSG3 and DSC2 were significantly correlated to the grade of differentiation, where decrease of these protein expression correlated to less differentiated phenotype. The expression of DSG1 or DSG2 was not significantly correlated to the grade of differentiation. The scores of DSG3 and DSC2 were significantly correlated to the pattern of invasion, where decrease of these protein expression correlated to more invasive tumor front. The expression of DSG1 or DSG2 was not significantly correlated to the pattern of invasion. Cancers positive for lymph node metastasis exhibited significantly lower scores of DSG3 and DSC2. The expression of DSG3 and DSC2 exhibited positive correlation to each other. BP180 and Laminin 332 expression showed less diversities among cases, mainly because their expression was restricted to the basal layer of the cancer nests. The expression of Laminin 332 was always cytoplasmic. The scores of BP180 and Laminin 332 were correlated to the grade of differentiation; cytoplasmic BP180 expression correlated to less differentiated histology and weak induction of Laminin 332 expression correlated to more differentiated histology. BP180 expression correlated to higher invasiveness and weak induction of Laminin 332 expression correlated to lower invasiveness. Discussion Similar to adherens junctions, desmosomes and hemidesmosomes comprise either cadherin- or integrin-family proteins to form junction complexes respectively. In desmosome adhesion, the two types of desmosomal cadherins; the desmogleins (DSG1 4) and the desmocollins (DSC1 3) bridge the neighboring cells through association of their ectodomains. The precise components of DSGs and DSCs varies in a tissue-specific manner. While malfunction of adherens junction can promote cancer development and metastasis, impaired desmosome function has not been associated with a predisposition to skin tumors. However, overexpression of desmosomal cadherins in squamous cell carcinoma cells, - 3 -

4 suppresses tumor formation and invasion in mouse xenograft models, suggesting their tumor-suppressive functions. It should be noted that DSG1 was not significantly linked to aggressive phenotypes of the oral cancer, although DSG1 was reduced in most cases. By contrast, decrease of the DSG3 expression as well as the DSC2 expression was significantly correlated to aggressive phenotypes of the oral cancer. This may be attributed to the fact that DSG3 was the dominant desmogleins in the basal cells of oral epithelium, where DSG1 expression was not clearly observed. The main role of Laminin 332 is to mediate static adhesion and hemidesmosome formation in normal tissues. Interestingly, when it is cleaved by matrix metalloproteinases, it stimulates cell migration and invasion.the cytoplasmic translocation of BP180 may indicate impaired assembly of hemidesmosomes or ongoing dynamic reorganization of hemidesmosomes. In line with this notion, cytoplasmic translocation of BP180 was associated with histological features showing less differentiation and higher invasiveness. Moreover, unaffected membranous localization of BP180 may warrant low probability of lymph node metastasis. The changes in the expression and localization of Laminin 332 or BP180 were not concomitant to each other, suggesting that these two hemidesmosome proteins can be used as independent markers to predict the cancer behaviors. In normal oral epithelium, DSG1, DSG3, DSC2 and BP180 were present on the plasma membrane. DSG3 was a more essential desmoglein than DSG1 in the basal layer. In oral cancer, the membranous presentation of these proteins reduced and exhibited a tendency of cytoplasmic accumulation. Moreover, ectopic expression of DSG2 and Laminin 332 was induced. The alterations of DSG3, DSC2, BP180 and Laminin 332 expression were linked to cancer behaviors such as the histological differentiation, the pattern of invasion and the presence of lymph node metastasis. Conclusion These results demonstrated the significance of desmosomal and hemidesmosomal proteins in generation of each oral cancer property. Combined use of these markers may prove useful for grading cancers and predicting the prognosis. < 和文による要約 > 種々の癌の分化や浸潤転移には細胞接着の異常が深く関与しているデスモソームとヘミデスモソームは細胞間および細胞と細胞外基質間の接着に関与する固定結合装置であるこれらが口腔癌の生物学的動態に及ぼす影響を明らかにするため 51 症例の口腔扁平上皮癌におけるデスモグレイン (DSG)1 DSG2 DSG3 デスモコリン(DSC)2 BP180 Laminin332 の発現を免疫組織化学的に検討した DSG1 DSG3 DSC2 は正常口腔上皮の細胞膜に局在する発現がみられた癌では細胞膜上の - 4 -

5 発現は減弱し細胞質への局在の変化を認めた BP180 は基底細胞の細胞膜に発現がみられた癌で BP180 は間質に相対する癌細胞に発現ししばしば細胞質への局在を認めたラミニン 332 の発現は正常上皮でみられなかった癌で Laminin332 は間質に相対する癌細胞に常に発現がみられた発現を準定量的に解析し組織学的分化度浸潤様式リンパ節転移の有無と比較した DSG3 と DSC2 の減弱は低分化と高浸潤性パターンと高頻度のリンパ節転移と有意に相関した BP180 の減少と Laminin332 の増加もこれらの高悪性の指標に相関した以上の結果から口腔癌の動態に与えるデスモソーム及びヘミデスモソームタンパクの重要性が示唆されたまたこれらのマーカーの使用は癌の悪性度評価と予後の予測に利用できる可能性が示唆された - 5 -

6 論文審査の要旨および担当者報告番号甲第 4768 号赵鑫論文審査担当者主査原田清副査高野吉郎原田浩之 ( 論文審査の要旨 ) 種々の癌の分化や浸潤転移には細胞接着の異常が深く関与しているデスモソームとヘミデスモソームは細胞間および細胞と細胞外基質間の接着に関与する固定結合装置である口腔癌におけるデスモソームとヘミデスモソームの発現の変化に関する報告は散見されるが未だそれらの結果には不一致があり全貌が曖昧であるまた各因子間の相関についても不明な点が多い赵はデスモソームとヘミデスモソームタンパクの変化が口腔癌の生物学的動態に及ぼす影響を明らかにするため 51 症例の口腔扁平上皮癌におけるデスモソーム構成タンパク (DSG1 DSG2 DSG3 DSC2) ヘミデスモソーム構成タンパク(ITGB4 COL17A1 LAMC2) の発現を免疫組織化学的に調べ正常組織と比較したさらに発現の変化と癌の分化度浸潤様式リンパ節転移の有無の相関を検討した本研究で得られた結果は以下の通りである 1.DSG1 DSG3 DSC2 の発現は正常口腔上皮の細胞膜に局在していた癌でこれらの膜上の発現は減弱し細胞質への局在の変化を認めた 2.DSG2 は正常上皮に比べて癌で発現が増強した 3.ITGB4 は正常上皮で基底膜にみられた癌では発現が増強し細胞質への局在の変化が見られた 4.COL17A1 は正常上皮基底細胞の細胞膜に見られた癌では間質に相対する癌細胞に発現ししばしば細胞質への局在の変化が見られた 5.LAMC2 は正常上皮で基底膜に見られた癌で LAMC2 は間質に相対する癌細胞の細胞質に常に発現が見られたが癌胞巣の基底膜相当部には認めなかった 6.DSG3 と DSC2 の細胞質への局在の変化および発現の減弱は低分化と高浸潤性パターンと高頻度のリンパ節転移と有意に相関した 7.COL17A1 の減少と LAMC2 の発現の増加は低分化と高浸潤性パターンと高頻度のリンパ節転移と相関した以上の結果から口腔癌の動態に及ぼすデスモソーム及びヘミデスモソームタンパクの重要性が示唆されこれらのマーカーが癌の悪性度評価に利用できる可能性が示唆された本研究から得られた知見は口腔癌の動態と悪性度の評価に有用であり今後の歯科医学および医学の進歩に大いに寄与するものと考えられ本論文は博士 ( 歯学 ) の学位を請求するのに十分価値あるものと認められた ( 1 )

Table 1. Numbers of Cancerous Pleurisy Cases According to Primary Site Table 2. Histological Type of Lung Carcinoma with Cancerous Pleurisy at Autopsy

Clinicopathological Studies on Cancerous Pleurisy, with Emphasis on Extra-Pulmonary Malignancy Table 1. Numbers of Cancerous Pleurisy Cases According to Primary Site Table 2. Histological Type of Lung