探討皮膚砷癌之表皮細胞異常增生及分化之分子調控機轉

Molecular mechanisms of abnormal epidermal proliferation and differentiation in arsenical skin cancers

呂建和(Jian-He Lu)

指導教授 ： 余幸司

高雄醫學大學/醫學院/醫學研究所博士班/博士(2018年)

摘要

台灣西南部沿海高砷污染地區常有區域性砷癌之發生。在砷暴露相關的癌症中，以皮膚癌最為常見，在砷引起的皮膚癌中，又以波文氏病 (一種表皮內癌) 居大多數。而人類表皮細胞動力平衡發生異常是造成皮膚病變的主因之一，此類病變若同時併發有-異生 (dysplasia)及異型性 (atypia) 的病理特徵變化，則可被定位為癌化。砷引起之人類表皮內癌的病理特性包含表皮異常增生 (acanthosis)、過度角化(keratosis)、角化異常 (dyskeratosis) 及異生 (dysplasia)。然而，砷導致波文氏病之癌化進展過程，從未在體外實驗室內發展成功。砷為化學致癌物。動物研究指出化學致癌作用由三個階段組成，包含啟動階段 (initiation)、促進階段 (promotion) 和發展階段 (progression)。然而，由於缺乏合適的多細胞構成的組織模型，因此涉及這些過程的細胞與細胞間相互作用的分子機制尚不清楚。目前我們已經利用人類角質細胞、纖維母細胞與周邊血單核細胞 (PBMC) 並在砷處理的條件下，建立了一種模擬人類表皮內癌皮膚組織培養模式。此種模擬人類表皮內癌皮膚組織培養模式能再現出砷誘導的波文氏病病理的形態學特徵，包括表皮異常增生 (acanthosis)、異生 (dysplasia) 和角化異常 (dyskeratosis) 等。因此，我們擬以此實驗模式進一步由基因及分子層面探討砷誘導皮膚癌過程中的病生理機轉。本研究論文探討: (I) 砷致表皮動態平衡異常及表皮內癌之過程中參與的基因及其調控；(II) 免疫細胞對砷引發表皮內癌過程中與表皮細胞間交互作用之功能性角色。
藉由此模擬組織培養模式，探討砷引發表皮癌化之進展過程，發現砷處理會增加表皮層的厚度，促進表皮細胞異常增生及增加鱗狀細胞之基底細胞增生指標cytokeratin 14 (CK14) 及調控表皮細胞增生之N-terminal truncated p63 isoform (ΔNp63) 基因的表現，角質細胞分化程度越高而表現量越高的分化指標cytokeratin 10 (CK10) 及調控表皮細胞分化之full-length p63 isoform (TAp63) 的表現量明顯降低。在砷處理的角質細胞處理組中，CK14及ΔNp63之表現量有增加，而CK10及TAp63之表現量則下降。藉由siRNA抑制ΔNp63基因之表現發現除了會抑制砷促進CK14表現的作用，亦會抑制砷降低TAp63與CK10之蛋白質表現的作用。在細胞模式中，我們證明砷造成的ΔNp63蛋白質表現量增加可能與波文氏病患者表皮異常增生有密切的關係。進一步利用模擬皮膚組織模式研究角質細胞與免疫細胞間交互作用對砷引發表皮內癌過程之作用機轉中，我們發現砷會刺激PBMC釋放腫瘤壞死因子-α (tumor necrosis factor-α) 而進一步促進表皮異常增生。
本論文主要是以皮膚砷癌中具代表性之波文氏病病理特徵為研究起點，並利用組織觀點進行砷引發表皮內癌過程中細胞間交互作用之分子機轉探討，所得結果可解釋皮膚砷癌波文氏病臨床與病理形態學；也期望本論文能對調控癌化過程與治療或預防提供之新的見解。

關鍵字

砷 ； 波文氏病 ； ΔNp63 ； TNF-α ； 表皮增生 ； 發炎作用

並列摘要

A high prevalent rate of cancers was noted in southwestern arsenism areas of Taiwan. Arsenic exposure has been demonstrated to induce human cancers. Among them, skin malignancy is one of the most commonly observed cancers. Bowen’s disease, an intraepidermal carcinoma in situ, is the main form of arsenic cancer in skin. Disruption of the epidermal cell dynamic homeostasis results in abnormal skin morphology, aberrant skin functions, and diseases. Furthermore, if dysplasia and atypia develop, a carcinogenic process is heralded. The characteristic morphologic changes including acanthosis, keratosis, dyskeratosis, and dysplasia are observed in epidermal carcinoma. Arsenic is a carcinogenic chemical. Animal studies have shown that chemical carcinogenesis consists of a three-stage process: initiation, promotion, and progression. However, because of the lack of a tissue model of arsenic carcinogenesis, the molecular mechanisms of cell-cell interactions involved in the dynamic process remain unclear. We have established a human intraepidermal carcinoma skin equivalent (SE) with organotypic culture - consisting of keratinocytes, fibroblasts, and peripheral blood mononuclear cells (PBMC) - induced by arsenic treatment. This SE reproduces the pathognomonic characteristics of arsenic-induced Bowen’s disease, including acanthosis, dysplasia, and dyskeratosis. Taking advantage of this innovative model, we set out to investigate the arsenic carcinogenesis through genomic and molecular approaches. The study aim to (Ⅰ) identify the chronological genetic focusing on disrupted epidermal homeostasis and de novo tumorigenesis of arsenic-induced intraepidermal carcinoma, and (Ⅱ) elucidate the functional roles of cellular interactions in the development of arsenic-induced intraepidermal carcinoma through immune cells mediated inflammation.
Using this SE model, we showed that arsenic increased epidermal thickness, induced abnormal proliferation, and increased expression of proliferating basal keratinocytes marker cytokeratin 14 (CK14) and N-terminal truncated p63 isoform (ΔNp63; proliferation regulator), whereas, that of the differentiation marker cytokeratin 10 (CK10) and full-length p63 isoform (TAp63; differentiation regulator) was decreased in squamous cells as compared with the control. Treatment of cultured normal human epidermal keratinocytes (HKCs) with arsenic increased CK14 and △Np63 expressions, but decreased TAp63 and CK10 expressions. Furthermore, knockdown of ΔNp63 expression by RNA interference abrogated arsenic-induced CK14 expression, and recovered the reduction of TAp63 and CK10 caused by arsenic. These findings indicated that ΔNp63 is a pivotal regulator in the abnormal cell proliferation in arsenical cancers. Furthermore, using this SE model to study the interaction between keratinocytes and immune cells in the process of arsenic-induced intraepithelial neoplasia, we showed that arsenic stimulated tumor necrosis factor-α release mainly from PBMC, which triggered abnormal epidermal proliferation.
Our data show that the cellular interaction between human keratinocytes and lymphocytes promotes the pathological characteristics of arsenic-induced Bowen's, and provide novel insights on arsenic-modulated a carcinogenic process.

並列關鍵字

Arsenic ； Bowen’s disease ； ΔNp63 ； TNF-α ； Epidermal proliferation ； Inflammation

參考文獻

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