摘要:過去的證據顯示角質細胞、纖維母細胞、血管細胞與腫瘤浸潤免疫細胞所形成的腫瘤微環境,是黑色素細胞癌發展與生成的關鍵。我們過去的研究證實 mal de Meleda症患者,係由於 SLURP-1蛋白在胺基酸第 86號位點由甘胺酸突變為精胺酸(G86R),所導致的一種罕見體染色體隱性遺傳的手足掌發紅角化症,這樣的患者具有容易罹患皮膚黑色素細胞癌的傾向。SLURP-1 蛋白,是一種乙醯膽鹼受體(nAChR)的變構配體,具有調節角質細胞增生、細胞凋亡和皮膚發炎反應的功能。此外,失去功能的突變 SLURP-1蛋白還造成了 mal de Meleda 的患者在 CD3/CD28 抗體刺激下 T 細胞活化功能的缺損。根據我們過去的這些研究發現,可能 mal de Meleda 患者的病灶提供了黑色素細胞癌易於生成並進展的微環境。我們假設這樣的黑色素細胞癌的形成及進展,可能是經由以下三種機轉:(1)角質細胞分泌生長因子至黑色素細胞癌微環境中;(2) T 細胞活化功能下降的免疫抑制微環境;(3)黑色素細胞癌分泌細胞激素或趨化素造成黑色素細胞癌微環境的免疫抑制,或癌細胞自身表現具有調節免疫功能之蛋白。 在這三年的計畫中,我們將使用 mal de Meleda 做為臨床研究模式系統,來探討黑色素細胞癌相關的皮膚微環境與 SLURP-1/nAChR 訊息傳遞路徑,著重於角質細胞與腫瘤浸潤免疫細胞的角色探討。在第一年的研究,我們將釐清在 mal de Meleda 對抗黑色素細胞癌的免疫微環境是經由何種機轉被抑制。我們首先將比較mal de Meleda患者與健康對照組周邊血液細胞中調節型(regulatory) T 細胞的比例。然後我們將剖析mal de Meleda患者 T細胞活化功能缺損的機轉,包括利用流式細胞儀分析表型標記、細胞介白素-2 (IL-2)的產生、鈣離子流量、相關的訊息傳遞路徑與活化 T 細胞的蛋白質體學。最後,將利用活體外的移行實驗與 NOD/SCID 小鼠動物模式,來探討 CD8+ 作用型(effector)與 CD25+ 調節型 T 細胞如何被 mal de Meleda 黑色素細胞癌所吸引。根據我們的初步發現,galectin-1 及 galectin-3的表現在 mal de Meleda 黑色素細胞癌細胞株有調控增加的現象,因此我們也會確立 galectin-1 及 galectin-3在mal de Meleda免疫調節的角色。在第二年的研究,我們將分析 mal de Meleda 患者的黑色素細胞癌的致癌基因突變。由於黑色素細胞癌細胞可能藉由產生具免疫調節作用的細胞激素或表現腫瘤抗原以製造一個免疫抑制的微環境來促進腫瘤發展,我們將利用我們已建立帶有 SLURP-1蛋白突變的mal de Meleda黑色素細胞癌細胞株來進行細胞激素的分析。在本研究的第三年,我們將試圖解析 SLURP-1 表現缺乏的角質細胞是否可以直接藉由通過細胞間相互作用或者間接經由旁分泌的方式促進黑色素細胞癌的增生、移行和入侵。 這個三年計畫之研究結果將可闡明 SLURP-1 蛋白在黑色素細胞癌相關的微環境,特別是角質細胞及免疫細胞所扮演的角色及功能,對黑色素細胞癌的重大意義。
Abstract: Evidence has amassed that keratinocytes, fibroblasts, vascular cells, and tumor infiltrating immune cells, which make up the tumor microenvironment, are essential for melanoma development and progression. Our previous works have demonstrated that patients with mal de Meleda (MDM), a rare autosomal recessive form of palmoplantar keratoderma caused by lymphocyte antigen 6/urokinase-type plasminogen activator receptor related protein-1 (SLURP-1) G86R mutation, are prone to develop cutaneous melanoma. SLURP-1, a positive allosteric ligand of 7-nicotinic acetylcholine receptors (nAChR), has been shown to regulate keratinocyte proliferation, apoptosis and cutaneous inflammation. Moreover, loss-of-function SLURP-1 mutation also caused impaired T-cell activation under anti-CD3/anti-CD28 stimulation in MDM patients. Based on these previous findings, we hypothesize that MDM provides an inflammatory microenvironment facilitating melanoma development, possibly through growth factor stimulation by keratinocytes in the immediate environment of melanocytes, or an immunosuppressive microenvironment that inhibit T cell activation. Tumors are also able to create an immunosuppressive microenvironment by producing cytokines/chemokines or expressing proteins functioning as immune regulator. In this 3-year proposal, we will use MDM as the feasible clinical model to investigate the interrelationship among melanoma-associated skin microenvironments and the SLURP-1/nAChR signaling pathway, focusing on the roles of keratinocytes and tumor-infiltrating immune cells. During the first year of the study, we will address the mechanisms how immune microenvironment against melanoma development is impaired in MDM. We will first compare the percentage of regulatory T (Treg) cells in peripheral blood mononuclear cells of MDM patients to the healthy controls. Then we will dissect the mechanisms of defective T cell activation in MDM patients, including flow cytometric analysis of phenotypic markers, IL-2 production, calcium influx, associated signaling transduction pathways, and proteomic analysis on stimulated T cells. Finally, the recruitment of CD8+ effector and CD25+ Treg cells into MDM melanoma cells will be investigated by in vitro migration assay and in vivo NOD/SCID mice model. We will also define the role of melanoma-associated galectin-1 and galectin-3, which are upregulated in MDM melanoma cell lines according to our preliminary data, as an immune regulator in MDM. During the second year of the study, we will first characterize the mutational profiling of oncogene drivers associated with melanomas in MDM. As tumor cells constantly orchestrate an immunosuppressive microenvironment by secreting immunoregulatory cytokines and presenting tumor antigens to promote tumor development, the cytokine profiling of the established MDM melanoma cell lines with SLURP-1 mutation will be analyzed. In the third year of the proposal, we will decipher whether keratinocytes with SLURP-1 knockdown can directly (through cell-cell interaction) or indirectly (through a paracrine fashion) promote melanoma proliferation, migration and invasion. Upon the completion of this 3-year proposal, important insights regarding the characterization and function of SLURP-1 in melanoma-associated microenvironments, addressing keratinocytes and immune surveillance, will be revealed.